Cancer diagnostic imaging agents

ABSTRACT

The present invention relates to a ligand-SIFA-chelator conjugate, comprising, within a single molecule three separate moieties: (a) one or more ligands which are capable of binding to PSMA, (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon atom and a fluorine atom, and (c) one or more chelating groups, containing a chelated nonradioactive cation.

The present invention relates to a ligand-SIFA-chelator conjugate,comprising, within a single molecule: (a) one or more ligands which arecapable of binding to PSMA, (b) a silicon-fluoride acceptor (SIFA)moiety which comprises a covalent bond between a silicon and a fluorineatom and which is labeled with ¹⁸F, and (c) one or more chelatinggroups, containing a chelated nonradioactive cation.

In this specification, a number of documents including patentapplications and manufacturer's manuals are cited. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all referenced documents are incorporated byreference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Prostate Cancer

Prostate Cancer (PCa) remained over the last decades the most commonmalignant disease in men with high incidence for poor survival rates.Due to its overexpression in prostate cancer, prostate-specific membraneantigen (PSMA) or glutamate carboxypeptidase II (GCP II) proved itseligibility as excellent target for the development of highly sensitiveradiolabelled agents for endoradiotherapy and imaging of PCa.Prostate-specific membrane antigen is an extracellular hydrolase whosecatalytic center comprises two zinc(II) ions with a bridging hydroxidoligand. It is highly upregulated in metastatic and hormone-refractoryprostate carcinomas, but its physiologic expression has also beenreported in kidneys, salivary glands, small intestine, brain and, to alow extent, also in healthy prostate tissue. In the intestine, PSMAfacilitates absorption of folate by conversion ofpteroylpoly-γ-glutamate to pteroylglutamate (folate). In the brain, ithydrolyses N-acetyl-Laspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartateand glutamate.

Prostate-Specific Membrane Antigen (PSMA)

Prostate-specific membrane antigen (PSMA) is a type II transmembraneglycoprotein that is highly overexpressed on prostate cancer epithelialcells. Despite its name, PSMA is also expressed, to varying degrees, inthe neovasculature of a wide variety of nonprostate cancers. Among themost common nonprostate cancers to demonstrate PSMA expression includebreast, lung, colorectal, and renal cell carcinoma.

The general necessary structures of PSMA targeting molecules comprise abinding unit that encompasses a zinc-binding group (such as urea,phosphinate or phosphoramidate) connected to a P1′ glutamate moiety,which warrants high affinity and specificity to PSMA and is typicallyfurther connected to an effector functionality. The effector part ismore flexible and to some extent tolerant towards structuralmodifications. The entrance tunnel accommodates two other prominentstructural features, which are important for ligand binding. The firstone is an arginine patch, a positively charged area at the wall of theentrance funnel and the mechanistic explanation for the preference ofnegatively charged functionalities at the P1 position of PSMA. Thisappears to be the reason for the preferable incorporation of negativecharged residues within the ligand-scaffold. An in-depth analysis aboutthe effect of positive charges on PSMA ligands has been, to ourknowledge, so far not conducted. Upon binding, the concertedrepositioning of the arginine side chains can lead to the opening of anS1 hydrophobic accessory pocket, the second important structure that hasbeen shown to accommodate an iodo-benzyl group of several urea basedinhibitors, thus contributing to their high affinity for PSMA.

Zhang et al. discovered a remote binding site of PSMA, which can beemployed for bidentate binding mode (Zhang et al., Journal of theAmerican Chemical Society 132, 12711-12716 (2010)). The so calledarene-binding site is a simple structural motif shaped by the sidechains of Arg463, Arg511 and Trp541, and is part of the GCPII entrancelid. The engagement of the arene binding site by a distal inhibitormoiety can result in a substantial increase in the inhibitor affinityfor PSMA due to avidity effects. PSMA I&T was developed with theintention to interact this way with PSMA, albeit no crystal structureanalysis of binding mode is available. A necessary feature according toZhang et al. is a linker unit (Suberic acid in the case of PSMA I&T)which facilitates an open conformation of the entrance lid of GCPII andthereby enabling the accessibility of the arene-binding site. It wasfurther shown that the structural composition of the linker has asignificant impact on the tumor-targeting and biologic activity as wellas on imaging contrast and pharmacokinetics, properties which arecrucial for both high imaging quality and efficient targetedendoradiotherapy.

Two categories of PSMA targeting inhibitors are currently used inclinical settings. On the one side there are tracers with chelatingunits for radionuclide complexation such as PSMA I&T or relatedcompounds. On the other side there are small molecules, comprising atargeting unit and effector molecules.

¹⁸F Labelling

Recently, several groups have focused on the development of novel¹⁸F-labelled urea-based inhibitors for PCa diagnosis. The ¹⁸F-labelledurea-based PSMA inhibitor ¹⁸F-DCFPyl demonstrated promising results inthe detection of primary and metastatic. Based on the structure ofPSMA-617, the ¹⁸F-labelled analogue PSMA-1007 was recently developed,which showed comparable tumor-to-organ ratios.

An attractive approach for introducing ¹⁸F labels is the use of siliconfluoride acceptors (SIFA). Silicon fluoride acceptors are described, forexample, in Lindner et al., Bioconjugate Chemistry 25, 738-749 (2014).In order to preserve the silicon-fluoride bond, the use of siliconfluoride acceptors introduces the necessity of sterically demandinggroups around the silicone atom. This in turn renders silicon fluorideacceptors highly hydrophobic. In terms of binding to the targetmolecule, in particular to the target molecule which is PSMA, thehydrophobic moiety provided by the silicone fluoride acceptor may beexploited for the purpose of establishing interactions of theradio-diagnostic or -therapeutic compound with the hydrophobic pocketdescribed in Zhang et al., Journal of the American Chemical Society 132,12711-12716 (2010). Yet, prior to binding, the higher degree oflipophilicity introduced into the molecule poses a severe problem withrespect to the development of radiopharmaceuticals with suitable in vivobiodistribution, i.e. low unspecific binding in non-target tissue.

Failure to Solve the Hydrophobicity Problem

Despite many attempts, the hydrophobicity problem caused by siliconfluoride acceptors has not been satisfactorily solved in the prior art.

To explain further, Schirrmacher E. et al. (Bioconjugate Chem. 2007, 18,2085-2089) synthesized different ¹⁸F-labelled peptides using the highlyeffective labelling synthon p-(di-tert-butylfluorosilyl) benzaldehyde([¹⁸F]SIFA-A), which is one example of a silicon fluoride acceptor. TheSIFA technique resulted in an unexpectedly efficient isotopic ¹⁹F—¹⁸Fexchange and yielded the ¹⁸F-synthon in almost quantitative yields inhigh specific activities between 225 and 680 GBq/μmol (6081-18 378Ci/mmol) without applying HPLC purification. [¹⁸F]SIFA-benzaldehyde wasfinally used to label the N-terminal amino-oxy (N-AO) derivatizedpeptides AO-Tyr3-octreotate (AO-TATE), cyclo(fK(AO-N)RGD) andN-AO-PEG₂-[D-Tyr-Gln-Trp-Ala-Val-Ala-His-Thi-Nle-NH₂] (AO-BZH3, abombesin derivative) in high radiochemical yields. Nevertheless, thelabelled peptides are highly lipophilic (as can be taken from the HPLCretention times using the conditions described in this paper) and thusare unsuitable for further evaluation in animal models or humans.

In Wängler C. et al. (Bioconjugate Chem., 2009, 20 (2), pp 317-321), thefirst SIFA-based Kit-like radio-fluorination of a protein (rat serumalbumin, RSA) has been described. As a labelling agent,4-(di-tert-butyl[¹⁸F]fluorosilyl)benzenethiol (Si[¹⁸F]FA-SH) wasproduced by simple isotopic exchange in 40-60% radiochemical yield (RCY)and coupled the product directly to maleimide derivatized serum albuminin an overall RCY of 12% within 20-30 min. The technically simplelabelling procedure does not require any elaborated purificationprocedures and is a straightforward example of a successful applicationof Si-18F chemistry for in vivo imaging with PET. The time-activitycureves and μPET images of mice showed that most of the activity waslocalized in the liver, thus demonstrating that the labelling agent istoo lipophilic and directs the in vivo probe to hepatobiliary excretionand extensive hepatic metabolism.

Wängler C. et al. (see Bioconjug Chem. 2010 Dec. 15; 21(12):2289-96)subsequently tried to overcome the major drawback of the SIFAtechnology, the high lipophilicity of the resultingradiopharmaceuticals, by synthesizing and evaluating new SIFA-octreotateanalogues (SIFA-Tyr3-octreotate, SIFA-Asn(AcNH-β-Glc)-Tyr3-octreotateand SIFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate). In these compounds,hydrophilic linkers and pharmacokinetic modifiers were introducedbetween the peptide and the SIFA-moiety, i.e. a carbohydrate and a PEGlinker plus a carbohydrate. As a measure of lipophilicity of theconjugates, the log P(ow) was determined and found to be 0.96 forSIFA-Asn(AcNH-β-Glc)-PEG-Tyr³-octreotate and 1.23 forSIFA-Asn(AcNH-β-Glc)-Tyr³-octreotate. These results show that the highlipophilicity of the SIFA moiety can only be marginally compensated byapplying hydrophilic moieties. A first imaging study demonstratedexcessive hepatic clearance/liver uptake and thus has never beentransferred into a first human study.

Bernard-Gauthier et al. (Biomed Res Int. 2014; 2014:454503) reviews agreat plethora of different SIFA species that have been reported in theliterature ranging from small prosthetic groups and other compounds oflow molecular weight to labelled peptides and most recently affibodymolecules. Based on these data the problem of lipophilicity ofSIFA-based prosthetric groups has not been solved sofar; i.e. amethodology that reduces the overall lipophilicity of a SIFA conjugatedpeptide to a log D lower than approx −2.0 has not been described.

In Lindner S. et al. (Bioconjug Chem. 2014 Apr. 16; 25(4):738-49) it isdescribed that PEGylated bombesin (PESIN) derivatives as specific GRPreceptor ligands and RGD (one-letter codes for arginine-glycine-asparticacid) peptides as specific αvβ3 binders were synthesized and tagged witha silicon-fluorine-acceptor (SIFA) moiety. To compensate the highlipophilicity of the SIFA moiety various hydrophilic structuremodifications were introduced leading to reduced logD values.SIFA-Asn(AcNH-β-Glc)-PESIN, SIFA-Ser(β-Lac)-PESIN, SIFA-Cya-PESIN,SIFA-LysMe3-PESIN, SIFA-γ-carboxy-d-Glu-PESIN, SIFA-Cya2-PESIN,SIFA-LysMe3-γ-carboxy-d-Glu-PESI N, SIFA-(γ-carboxy-d-Glu)2-PESIN,SIFA-RGD, SIFA-γ-carboxy-d-Glu-RGD, SIFA-(γ-carboxy-d-Glu)2-RGD,SIFA-LysMe3-γ-carboxy-d-Glu-RGD. All of these peptides—already improvedand derivatized with the aim to reduce the lipophilicity—showed a logDvalue in the range between +2 and −1.22.

In view of the above, the technical problem underlying the presentinvention can be seen in providing radio-diagnostics which contain asilicone fluoride acceptor and which are, at the same time,characterized by favourable in-vivo properties.

As will be become apparent in the following, the present inventionestablished a proof-of-principle using specific conjugates which bindwith high affinity to prostate-specific antigen (PSMA) as target.Accordingly, a further technical problem underlying the presentinvention can be seen in providing improved diagnostics for the medicalindication which is cancer, preferably prostate cancer.

These technical problems are solved by the subject-matter of the claims.Accordingly, in the first aspect, the present invention relates to aligand-SIFA-chelator conjugate, comprising, within a single molecule:(a) one or more ligands which are capable of binding to PSMA, (b) asilicon-fluoride acceptor (SIFA) moiety which comprises a covalent bondbetween a silicon and a fluorine atom and which is labeled with ¹⁸F, and(c) one or more chelating groups, containing a chelated nonradioactivecation.

The ligand-SIFA-chelator conjugate comprises three separate moieties.The three separate moieties are a) one or more ligands which are capableof binding to PSMA, (b) a silicon-fluoride acceptor (SIFA) moiety whichcomprises a covalent bond between a silicon and a fluorine atom, and (c)one or more chelating groups, containing a chelated nonradioactivecation.

For diagnostic imaging, the fluorine atom on the SIFA moiety is ¹⁸F. The¹⁸F can be introduced by isotopic exchange with ¹⁹F.

Whilst certain ligands which are capable of binding to adisease-relevant target molecule may be cyclic peptides, such cyclicpeptides are not chelating groups as envisaged herein, as the problem ofthe hydrophobic SIFA moiety is not solved in the absence of a furtherchelating moiety. Thus compounds of the invention require a hydrophilicchelating group in addition to the ligands which are capable of bindingto PSMA. The hydrophilic chelating group is required to reduce thehydrophobic nature of the compounds caused by the presence of the SIFAmoiety.

The ligand in relation to the first aspect of the invention is definedin functional terms. This is the case because the present invention doesnot depend on the specific nature of the ligand in structural terms.Rather, a key aspect of the invention is the combination, within asingle molecule, of a silicon fluoride acceptor and a chelator or achelate. These two structural elements, SIFA and the chelator, exhibit aspatial proximity. Preferably, the shortest distance between two atomsof the two elements is less or equal 25 Å, more preferably less than 20Å and even more preferably less than 15 Å. Alternatively or in addition,it is preferred that not more than 25 covalent bonds separate an atom ofthe SIFA moiety and an atom the chelator, preferably not more than 20chemical bonds and even more preferably not more than 15 chemical bonds.

The cation in accordance with item (c) of the first aspect is anon-radioactive cation. It is preferably a non-radioactive metal cation.Examples are given further below.

As a consequence, conjugates fall under the terms of the first aspectwhich are radioactively labelled at the SIFA moiety, as well asmolecules which are not radiolabelled at all. In the latter case, thechelating group may be either a complex of a cold (non-radioactive) ionor may be devoid of any ion.

The present inventors surprisingly discovered that placement of thesilicone fluoride acceptor in the neighbourhood of a hydrophilicchelator such as, but not limited to, DOTAGA or DOTA, shields orcompensates efficiently the lipophilicity of the SIFA moiety to anextent which shifts the overall hydrophobicity of compound in a rangewhich renders the compound suitable for in-vivo administration.

A further advantage of the compounds, especially of PSMA targetedcompounds of the present invention is their surprisingly lowaccumulation in the kidneys of mice when compared to other PSMA targetedradiopharmaceuticals, such as PSMA I&T. Without wishing to be bound by aparticular theory, it seems to be the combination of the structuralelement SIFA with a chelator which provides for the unexpected reductionof accumulation in the kidneys.

In terms of lipophilicity/hydrophilicity, the logP value (sometimes alsoreferred to as logD value) is an art-established measure.

The term “lipophilicity” relates to the strength of being dissolved in,or be absorbed in lipid solutions, or being adsorbed at a lipid-likesurface or matrix. It denotes a preference for lipids (literal meaning)or for organic or apolar liquids or for liquids, solutions or surfaceswith a small dipole moment as compared to water. The term“hydrophobicity” is used with equivalent meaning herein. The adjectiveslipophilic and hydrophobic are used with corresponding meaning to thesubstantives described above.

The mass flux of a molecule at the interface of two immiscible orsubstantially immiscible solvents is governed by its lipophilicity. Themore lipophilic a molecule is, the more soluble it is in the lipophilicorganic phase. The partition coefficient of a molecule that is observedbetween water and n-octanol has been adopted as the standard measure oflipophilicity. The partition coefficient P of a species A is defined asthe ratio P=[A]_(n-octanol)/[A]_(water). A figure commonly reported isthe logP value, which is the logarithm of the partition coefficient. Incase a molecule is ionizable, a plurality of distinct microspecies(ionized and not ionized forms of the molecule) will in principle bepresent in both phases. The quantity describing the overalllipophilicity of an ionizable species is the distribution coefficient D,defined as the ratio D=[sum of the concentrations of allmicrospecies]_(n-octanol)/[sum of the concentrations of allmicrospecies]_(water). Analogous to logP, frequently the logarithm ofthe distribution coefficient, logD, is reported. Often, a buffer system,such as phosphate buffered saline is used as alternative to water in theabove described determination of logP.

If the lipophilic character of a substituent on a first molecule is tobe assessed and/or to be determined quantitatively, one may assess asecond molecule corresponding to that substituent, wherein said secondmolecule is obtained, for example, by breaking the bond connecting saidsubstituent to the remainder of the first molecule and connecting (the)free valence(s) obtained thereby to hydrogen(s).

Alternatively, the contribution of the substituent to the logP of amolecule may be determined. The contribution π_(X x) of a substituent Xto the logP of a molecule R—X is defined asπ_(X x)=logP_(R—X)−logP_(R—H), wherein R—H is the unsubstituted parentcompound.

Values of P and D greater than one as well as logP, logD and π_(X x)values greater than zero indicate lipophilic/hydrophobic character,whereas values of P and D smaller than one as well as logP, logD andπ_(X x) values smaller than zero indicate hydrophilic character of therespective molecules or substituents.

The above described parameters characterizing the lipophilicity of thelipophilic group or the entire molecule according to the invention canbe determined by experimental means and/or predicted by computationalmethods known in the art (see for example Sangster, Octanol-waterPartition Coefficients: fundamentals and physical chemistry, John Wiley& Sons, Chichester. (1997)).

In a preferred embodiment, the logP value of the compounds of theinvention is between −5 and −1.5. It is particularly preferred that thelogP value is between −3.5 and −2.0.

In a preferred embodiment, a ligand in accordance with the inventioncomprises or consists of a peptide, a peptidomimetic or a substitutedurea, substituents including amino acids. It is understood that a ligandwhich comprises a peptide or peptidomimetic also comprises anon-peptidic and non-peptidomimetic part. In terms of molecular weight,preference is given to molecular weights below 15 kDa, below 10 kDa orbelow 5 kDa. Accordingly, small proteins are also embraced by the term“ligand”. Target molecules are not particularly limited and includeenzymes, receptors, epitopes, transporters, cell surface molecules andproteins of the extracellular matrix. Preferred are targets which aredisease relevant. Particularly preferred are targets which are causallyinvolved in a given disease, or which are highly overexpressed in agiven disease and/or the inhibition of which can cause a beneficialeffect in a patient suffering from a given disease. The ligands arepreferably high affinity ligands with preferable affinity, expressed asIC₅₀, being below 50 nM, below 20 nM or below 5 nM.

Especially preferred are those ligands which bind with high affinity toprostate-specific membrane antigen (PSMA).

Preferably, the silicon-fluoride acceptor (SIFA) moiety has thestructure represented by formula (I):

Wherein F is understood to encompass both ¹⁹F and ¹⁸F, R^(1S) and R^(2S)are independently a linear or branched C3 to C10 alkyl group, preferablyR^(1S) and R^(2S) are selected from isopropyl and tert-butyl, and aremore preferably R^(1S) and R^(2S) are tert-butyl; R^(3S) is a C1 to C20hydrocarbon group which may comprise one or more aromatic and one ormore aliphatic units and/or up to 3 heteroatoms selected from O and S,preferably R^(3S) is a C6 to C10 hydrocarbon group which comprises anaromatic ring and which may comprise one or more aliphatic units; morepreferably R^(3S) is a phenyl ring, and most preferably, R^(3S) is aphenyl ring wherein the Si-containing substituent and the bond marked by

are in a para-position, and wherein the SIFA moiety is attached to theremainder of the conjugate via the bond marked by

.

More preferably, the silicon-fluoride acceptor (SIFA) moiety has thestructure represented by formula (Ia):

wherein t-Bu indicates a tert-butyl group;

and F is understood to encompass both ¹⁹F and ¹⁸F

A preferred chelating group comprises at least one of the following (i),(ii) or (iii).

(i) A macrocyclic ring structure with 8 to 20 ring atoms of which 2 ormore, more preferably 3 or more, are selected from oxygen atoms ornitrogen atoms. Preferably, 6 or less ring atoms are selected fromoxygen atoms or nitrogen atoms. Especially preferred is that 3 or 4 ringatoms are nitrogen atoms or oxygen atoms. Among the oxygen and nitrogenatoms, preference is given to the nitrogen atoms. In combination withthe macrocyclic ring structure, the preferred chelating group maycomprise 2 or more, such as 2 to 6, preferably 2 to 4, carboxyl groupsand/or hydroxyl groups. Among the carboxyl groups and the hydroxylgroups, preference is given to the carboxyl groups.

(ii) An acyclic, open chain chelating structure with 8 to 20 main chain(back bone) atoms of which 2 or more, more preferably 3 or more areheteroatoms selected from oxygen atoms or nitrogen atoms. Preferably, 6or less back bone atoms are selected from oxygen atoms or nitrogenatoms. Among the oxygen and nitrogen atoms, preference is given to thenitrogen atoms. More preferably, the open chain chelating structure is astructure which comprises a combination of 2 or more, more preferably 3or more heteroatoms selected from oxygen atoms or nitrogen atoms, and 2or more, such as 2 to 6, preferably 2 to 4, carboxyl groups and/orhydroxyl groups. Among the carboxyl groups and the hydroxyl groups,preference is given to the carboxyl groups.

(iii) A branched chelating structure containing a quaternary carbonatom. Preferably the quaternary carbon atom is substituted with 3identical chelating groups in addition to the SIFA/ligand moiety. Thesubstituted chelating groups can comprise an amide. The substitutedchelating groups can comprise an aromatic group. The substitutedchelating groups can comprise a hydroxypyridinone.

In preferred specific examples, the chelating group is a residue of achelating agent selected frombis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a),cyclohexyl-1,2-diaminetetraacetic acid (CDTA),4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA),N′-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide(DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan(DO2A) 1,4,7,10-tetracyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA),α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTAGA), 1,4,7,10 tetraazacyclododecane N,N′,N″,N′″1,4,7,10-tetra(methylene) phosphonic acid (DOTMP),N,N′-dipyridoxylethylendiamine-N,N′-diacetate-5,5′-bis(phosphat) (DPDP),diethylene triamine N,N′,N″ penta(methylene) phosphonic acid (DTMP),diethylenetriaminepentaacetic acid (DTPA),ethylenediamine-N,N′-tetraacetic acid (EDTA),ethyleneglykol-O,O-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA),N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED),hydroxyethyldiaminetriacetic acid (HEDTA),1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate(HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), tetra3-hydroxy-N-methyl-2-pyridinone chelators(4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoicacid), abbreviated as Me-3,2-HOPO, 1,4,7-triazacyclononan-1-succinicacid-4,7-diacetic acid (NODASA),1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7-triazacyclononane(NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA),4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane(TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA),tris(hydroxypyridinone) (THP), terpyridin-bis(methyleneamintetraaceticacid (TMT),1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinicacid] (TRAP), 1,4,7,10-tetraazacyclotridecan-N,N′,N″,N′″-tetraaceticacid (TRITA),3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazonan-1-yl]methyl-hydroxy-phosphoryl]propanoicacid, and triethylenetetraaminehexaacetic acid (TTHA), which residue isprovided by covalently binding a carboxyl group contained in thechelating agent to the remainder of the conjugate via an ester or anamide bond.

Particular chelators are shown below:

Among the above exemplary chelating agents, particular preference isgiven to a chelating agent selected from TRAP, DOTA and DOTAGA.

Metal- or cation-chelating macrocyclic and acyclic compounds arewell-known in the art and available from a number of manufacturers.While the chelating moiety in accordance with the present invention isnot particularly limited, it is understood that numerous moieties can beused in an off-the-shelf manner by a skilled person without further ado.

The chelating group may comprise a chelated cation which isnon-radioactive.

Preferred examples of cations that may be chelated by the chelatinggroup are the non-radioactive cations of Sc, Cr, Mn, Co, Fe, Ni, Cu, Ga,Zr, Y, Tc, Ru, Rh, Pd, Ag, In, Sn, te, Pr, Pm, Tb, Sm, Gd, Tb, Ho, Dy,Er, Yb, Tm, Lu, Re, Pt, Hg, Au, Pb At, Bi, Ra, Ac, Th; more preferablythe cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Th and Er.The cation may be Ga. The cation may be Lu.

Accordingly, the ligand is preferably capable of binding toprostate-specific membrane antigen (PSMA).

More preferably, the ligand has the structure represented by formula(II):

wherein m is an integer of 2 to 6, preferably 2 to 4, more preferably 2;n is an integer of 2 to 6, preferably 2 to 4, more preferably 2 or 3;R^(1L) is CH₂, NH or O, preferably NH; R^(3L) is CH₂, NH or O,preferably NH; R^(2L) is C or P(OH), preferably C; and wherein theligand is attached to the remainder of the conjugate via the bond markedby

.

The ligand can have the structure represented by formula (IIa):

wherein n is an integer of 2 to 6; and wherein the ligand is attached tothe remainder of the conjugate via the bond marked by

.

A number of PSMA binders are known in the art which are all suitable inaccordance with the invention. The above preferred embodiment is astructural definition of a preferred group of PSMA binders.

It is particularly preferred that the conjugate of the first aspect is acompound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

SIFA is a silicon-fluoride acceptor (SIFA) moiety which comprises acovalent bond between a silicon and a fluorine atom and which is labeledwith ¹⁸F; preferably SIFA is the SIFA moiety of formula (I) and morepreferably of formula (Ia) defined above;

m is an integer of 2 to 6, preferably 2 or 3, more preferably 2;

n is an integer of 2 to 6, preferably 2 or 3, more preferably 2 or 4;

R^(1L) is CH₂, NH or O, preferably NH;

R^(3L) is CH₂, NH or O, preferably NH;

R^(2L) is C or P(OH), preferably C;

X¹ is selected from an amide bond, an ether bond, a thioether bond, anester bond, a thioester bond, an urea bridge, and an amine bond,preferably an amide bond;

X² is selected from an amide bond, an ether bond, a thioether bond, anester bond, a thioester bond, an urea bridge, and an amine bond,preferably an amide bond;

L¹ is a divalent linking group with a structure selected from anoligoamide, an oligoether, an oligothioether, an oligoester, anoligothioester, an oligourea, an oligo(ether-amide), anoligo(thioether-amide), an oligo(ester-amide), anoligo(thioester-amide), oligo(urea-amide), an oligo(ether-thioether), anoligo(ether-ester), an oligo(ether-thioester), an oligo ether-urea), anoligo(thioether-ester), an oligo(thioether-thioester), anoligo(thioether-urea), an oligo(ester-thioester), an oligo(ester-urea),and an oligo(thioester-urea), preferably with a structure selected froman oligoamide and an oligo(ester-amide).

L¹ can be optionally substituted with one or more substituentsindependently selected from —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, and—NHC(NH)NH₂.

X³ is selected from an amide bond, and ester bond, an ether, an amine,and a linking group of the formula:

wherein the bond marked by

at the NH group is bound to R^(B) and the other bond marked by

is bound to SIFA; preferably X³ is an amide bond; R^(B) is a trivalentcoupling group.

X⁴ is selected from an amide bond, an ether bond, a thioether bond, andester bond, a thioester bond, a urea bridge, an amine bond, a linkinggroup of the formula:

wherein the amide bond marked by

is formed with the chelating group, and the other bond marked by

is bound to R^(B); and a linking group of the formula:

wherein the bond marked by

at the carbonyl end is formed with the chelating group, and the otherbond marked by

is bound to R^(B); preferably X⁴ is an amide bond.

R^(CH) is chelating group containing a chelated nonradioactive cation,preferably a nonradioactive metal cation, wherein preferred embodimentsof said chelating group and of the optional chelated cation are asdefined above.

The term “oligo” as used in oligoamide, oligoether, oligothioether,oligoester, oligothioester, oligourea, oligo(ether-amide),oligo(thioether-amide), oligo(ester-amide), oligo(thioester-amide),oligo(urea-amide), oligo(ether-thioether), oligo(ether-ester),oligo(ether-thioester), oligo (ether-urea), oligo(thioether-ester),oligo(thioether-thioester), oligo(thioether-urea),oligo(ester-thioester), oligo(ester-urea), and oligo(thioester-urea) ispreferably to be understood as referring to a group wherein 2 to 20,more preferably wherein 2 to 10 subunits are linked by the type of bondsspecified in the same terms. As will be understood by the skilledreader, where two different types of bonds are indicated in brackets,both types of bonds are contained in the concerned group (e.g. in “oligo(ester-amide)”, ester bonds and amide bonds are contained).

It is preferred that L¹ comprises a total of 1 to 5, more preferably atotal of 1 to 3, and most preferably a total of 1 or 2 amide and/orester bonds, preferably amide bonds, within its backbone.

The term oligoamide therefore describes a moiety having a chain of CH₂or CHR groups interspersed with groups selected from NHCO or CONH. Eachoccurrence of the R moiety is an optional substituent selected from —OH,—OCH₃, —COOH, —COOCH₃, —NH₂, and —NHC(NH)NH₂.

It is also preferred that —X¹-L¹-X²— represents one of the followingstructures (L-1) and (L-2):

—NH—C(O)—R⁶—C(O)—NH—R⁷—NH—C(O)—  (L-1)

—C(O)—NH—R⁸—NH—C(O)—R⁹—C(O)—NH—R¹⁰—NH—C(O)—  (L-2)

wherein R⁶ to R¹⁰ are independently selected from C2 to C10 alkylene,preferably linear C2 to C10 alkylene, which alkylene groups may each besubstituted by one or more substitutents independently selected from—OH, —OCH₃, —COOH, —COOCH₃, —NH₂, and —NHC(NH)NH₂.

Especially preferred is that the total number of carbon atoms in R⁶ andR⁷ is 4 to 20, more preferably 4 to 16, without carbon atoms containedin optional substituents. Especially preferred is that the total numberof carbon atoms in R⁸ to R¹⁰, is 6 to 20, more preferably 6 to 16,without carbon atoms contained in optional substituents.

It is particularly preferred that —X¹-L¹-X²— represents one of thefollowing structures (L-3) and (L-4):

—NH—C(O)—R¹¹—C(O)—NH—R¹²—CH(COOH)—NH—C(O)—  (L-3)

—C(O)—NH—CH(COOH)—R¹³—NH—C(O)—R¹⁴—C(O)—NH—R¹⁵—CH(COOH)—NH—C(O)—  (L-4)

wherein R¹¹ to R¹⁵ are independently selected from C2 to C8 alkylene,preferably linear C2 to C8 alkylene.

Especially preferred is that the total number of carbon atoms in R¹¹ andR¹² or R¹³ to R¹⁵, respectively, is 8 to 18, more preferably 8 to 12,yet more preferably 9 or 10.

Preferably, R^(B) has the structure represented by formula (IV):

wherein: A is selected from N, CR¹⁶, wherein R¹⁶ is H or C1-C6 alkyl,and a 5 to 7 membered carbocyclic or heterocyclic group; preferably A isselected from N and CH, and more preferably A is CH; the bond marked by

at (CH₂)_(a) is formed with X², and a is an integer of 0 to 4,preferably 0 or 1, and most preferably 0; the bond marked by

at (CH₂)_(b) is formed with X³, and b is an integer of 0 to 4,preferably of 0 to 2, and more preferably 0 or 1; and the bond marked by

at (CH₂)_(c) is formed with X⁴, and c is an integer of 0 to 4,preferably of 0 to 2, and more preferably 0 or 1.

Even more preferred as a conjugate in accordance with the invention is acompound of formula (IIIa):

or a pharmaceutically acceptable salt thereof, wherein m, n, R^(1L),R^(2L), R^(3L), X¹, L¹, b, c, X⁴ and R^(CH) are as defined above,including all preferred embodiments thereof.

It is preferred for the compound of formula (IIIa) that b+c≥1.

It is also preferred for the compound of formula (IIIa) that b+c≤3.

It is more preferred for the compound of formula (IIIa) that b is 1 andc is 0.

It is also preferred for the compound of formula (III) that —X⁴—R^(CH)represents a residue of a chelating agent selected from DOTA and DOTAGAbound with one of its carboxylic groups via an amide bond to theremainder of the conjugate.

In a preferred embodiment of the compound of formula (III), saidcompound is a compound of formula (IIIb):

or a pharmaceutically acceptable salt thereof, wherein m, n, R^(1L),R^(2L), R^(3L), X¹, L¹, b, c, X⁴ and R^(CH) are as defined above; and ris 0 or 1.

Especially preferred is that —N(H)—R^(CH) represents a residue of achelating agent selected from DOTA and DOTAGA bound with one of itscarboxylic groups via an amide bond to the remainder of the conjugate.

In order to be used in PET imaging, the compounds require a positronemitting atom. The compounds include ¹⁸F for medical use. Most preferredcompounds of the invention are wherein F includes ¹⁸F and M³⁺ refers toa nonradioactive metal cation.

In the compounds herein, the nonradioactive metal cation M may bechelated to one or more COO⁻ groups. M may be chelated to one or more Natoms. M may be chelated to one or more N atoms or one or more COO⁻groups. M may be chelated to one or more N atoms and one or more COO⁻groups. Where the chelated nonradioactive metal cation M is shown, theacid groups to which it is chelated are merely representatively shown asCOO⁻, the equivalent fourth acid may also be partly chelated and hencemay not literally be COOH.

PSMA-SIFA1 (5)

and isomers thereof:

PSMA-SIFA2 (6)

and isomers thereof

PSMA-SIFA3 (7)

and isomers thereof

PSMA-SIFA4 (8)

and isomers thereof

PSMA-SIFA5 (9)

and isomers thereof

PSMA-SIFA 10

and isomers thereof:

PSMA-SIFA 11

and isomers thereof:

Preferred labelling schemes for these most preferred compounds are asdefined herein above.

In a further aspect, the present invention provides a pharmaceuticalimaging composition comprising or consisting of one or more conjugatesor compounds of the invention as disclosed herein above.

In a further aspect, the present invention provides a diagnosticcomposition comprising or consisting of one or more conjugates orcompounds of the invention as disclosed herein above.

The pharmaceutical composition may further comprise pharmaceuticallyacceptable carriers, excipients and/or diluents. Examples of suitablepharmaceutical carriers, excipients and/or diluents are well known inthe art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc.

Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, topical,intradermal, intranasal or intrabronchial administration. It isparticularly preferred that said administration is carried out byinjection and/or delivery, e.g., to a site in the pancreas or into abrain artery or directly into brain tissue. The compositions may also beadministered directly to the target site, e.g., by biolistic delivery toan external or internal target site, like the pancreas or brain. Thedosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Pharmaceutically active matter may be presentin amounts between 0.1 ng and 10 mg/kg body weight per dose; however,doses below or above this exemplary range are envisioned, especiallyconsidering the aforementioned factors.

In a further aspect, the present invention provides one or morecompounds of the invention as disclosed herein above for use indiagnostic medicine.

Preferred uses in medicine are in nuclear medicine such as nucleardiagnostic imaging, also named nuclear molecular imaging, and/ortargeted radiotherapy of diseases associated with an overexpression,preferably of PSMA on the diseased tissue.

In a further aspect, the present invention provides a compound of theinvention as defined herein above for use in a method of diagnosingand/or staging cancer, preferably prostate cancer. Prostate cancer isnot the only cancer to express PSMA. Nonprostate cancers to demonstratePSMA expression include breast, lung, colorectal, and renal cellcarcinoma. Thus any compound described herein having a PSMA bindingmoiety can be used in the diagnosis, imaging or treatment of a cancerhaving PSMA expression.

Preferred indications are the detection or staging of cancer, such as,but not limited high grade gliomas, lung cancer and especially prostatecancer and metastasized prostate cancer, the detection of metastaticdisease in patients with primary prostate cancer of intermediate-risk tohigh-risk, and the detection of metastatic sites, even at low serum PSAvalues in patients with biochemically recurrent prostate cancer. Anotherpreferred indication is the imaging and visualization of neoangiogensis.

In a further aspect, the present invention provides a conjugate orcompound of the invention as defined herein above for use in a method ofdiagnosing and/or staging cancer, preferably prostate cancer.

The Figures illustrate:

FIG. 1: Exemplary correlation of determination of the nine referencesubstances in OriginPro 2016G.

FIG. 2a : Quality Control of [19F][natGa]-rhPSMA7-rac([19F][natGa]D/L-Dap-R/S-DOTAGA-rhPSMA-7-rac), (batch 10, precursor forthe production of ([18F][natGa]D/L-Dap-R/S-DOTAGA-rhPSMA-7 at theDepartment of Nuclear Medicine, TUM). HPLC-conditions: Solvent A:H20+0.1% TFA; Solvent B: MeCN+0.1% TFA. Gradient: 25-35% B 0-40 min,95-95% B 40-45 min, 35-35% B 45-50 min; flow: 1 mL/min, column:Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM (DMSO), 10 μL

FIG. 2b : Peak assignment: D-Dap-R-DOTAGA-rhPSMA-7.1 rhPSM7-rac fromFIG. 2a coinjected with enantiopure D-Dap-R-DOTAGA-rhPSMA-7.1.HPLC-conditions: Solvent A: H20+0.1% TFA; Solvent B: MeCN+0.1% TFA.Gradient: 25-35% B 0-40 min, 95-95% B 40-45 min, 35-35% B 45-50 min;flow: 1 mL/min, column: Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM(DMSO), 10 μL

FIG. 2c : HPLC profile of D-Dap-R-DOTAGA-rhPSMA-7.1. HPLC-conditions:Solvent A: H20+0.1% TFA; Solvent B: MeCN+0.1% TFA. Gradient: 25-35% B0-40 min, 95-95% B 40-45 min, 35-35% B 45-50 min; flow: 1 mL/min,column: Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM (DMSO), 10 μL

FIG. 3a : Peak assignment: L-Dap-R-DOTAGA-rhPSMA-7.2 rhPSM7-rac fromFIG. 2a coinjected with enantiopure L-Dap-R-DOTAGA-rhPSMA-7.2.HPLC-conditions: Solvent A: H20+0.1% TFA; Solvent B: MeCN+0.1% TFA.Gradient: 25-35% B 0-40 min, 95-95% B 40-45 min, 35-35% B 45-50 min;flow: 1 mL/min, column: Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM(DMSO), 10 μL

FIG. 3b : HPLC profile of L-Dap-R-DOTAGA-rhPSMA-7.2 HPLC-conditions:Solvent A: H20+0.1% TFA; Solvent B: MeCN+0.1% TFA. Gradient: 25-35% B0-40 min, 95-95% B 40-45 min, 35-35% B 45-50 min; flow: 1 mL/min,column: Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM (DMSO), 10 μL

FIG. 4a : Peak assignment: D-Dap-S-DOTAGA-rhPSMA-7.3 rhPSM7-rac fromFIG. 2a coinjected with enantiopure D-Dap-S-DOTAGA-rhPSMA-7.3.HPLC-conditions: Solvent A: H20+0.1% TFA; Solvent B: MeCN+0.1% TFA.Gradient: 25-35% B 0-40 min, 95-95% B 40-45 min, 35-35% B 45-50 min;flow: 1 mL/min, column: Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM(DMSO), 10 μL

FIG. 4b : HPLC profile of D-Dap-D-DOTAGA-rhPSMA-7.3 HPLC-conditions:Solvent A: H20+0.1% TFA; Solvent B: MeCN+0.1% TFA. Gradient: 25-35% B0-40 min, 95-95% B 40-45 min, 35-35% B 45-50 min; flow: 1 mL/min,column: Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM (DMSO), 10 μL

FIG. 5a : Peak assignment: L-Dap-S-DOTAGA-rhPSMA-7.4 rhPSM7-rac fromFIG. 2a coinjected with enantiopure D-Dap-S-DOTAGA-rhPSMA-7.3.HPLC-conditions: Solvent A: H20+0.1% TFA; Solvent B: MeCN+0.1% TFA.Gradient: 25-35% B 0-40 min, 95-95% B 40-45 min, 35-35% B 45-50 min;flow: 1 mL/min, column: Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM(DMSO), 10 μL

FIG. 5b : HPLC profile of L-Dap-D-DOTAGA-rhPSMA-7.4 HPLC-conditions:Solvent A: H20+0.1% TFA; Solvent B: MeCN+0.1% TFA. Gradient: 25-35% B0-40 min, 95-95% B 40-45 min, 35-35% B 45-50 min; flow: 1 mL/min,column: Nucleosil 100-5 C18, 125×4.6 mm, Sample: 1 mM (DMSO), 10 μL

FIG. 6a : Binding affinities (IC50 [nM]) of rhPSMA7.1 and 7.2 to PSMA.Affinities were determined using LNCaP cells (150000 cells/well) and((4-[125I]iodobenzoyl)KuE ([125I]IB-KuE; c=0.2 nM) as the radioligand (1h, 4° C., HBSS+1% BSA). Data are expressed as mean±SD (n=3, in 3different experiments).

FIG. 6b : Determination of the binding affinities [nM] of the rhPSMA7isomers to PSMA. Each of the four column shows the individual affinitymeasurements for rhPSAM7.1 (left) to rhPSMA7.4 (right). Conditions asdescribed in the legend to FIG. 6 a.

FIG. 7: Depiction of the individual IC50 [nM] measurements shown inFIGS. 6a and 6b . Value No 5 of rhPSMA7.1 was deleted Conditions asdescribed in the legend to FIG. 6a

FIG. 8: Depiction of the individual internalization measurements [% of[¹²⁵I]IB-KuE]. Internalized activity (c=0.5 nM) at 1 hour as % of thereference ligand ([¹²⁵I]I-BA)KuE (c=0.2 nM), determined on LNCaP cells(37° C., DMEM F12+5% BSA, 125000 cells/well). Data is corrected fornon-specific binding (10 μmol PMPA) and expressed as mean±SD (n=3).

FIG. 9: Depiction of the individual measurements of the logP's of therhPSMA isomers.

FIG. 10: Biodistribution (in % ID/g) of ¹⁸F-labeled rhPSMA tracers at 1h p.i in LNCaP tumor-bearing SCID mice. Data are expressed as mean±SD(n=4 for rhPSMA7.1, n=5 for 7.2, n=4 for 7.3, n=5 for 7.4 and n=3 for7-rac).

FIG. 11: Biodistribution [% ID/g] of ¹⁸F-rhPSMAs co-injected with PMPA(8 mg/kg) at 1 h p.i in LNCaP tumor-bearing SCID mice. Data areexpressed as mean±SD (n=3).

FIG. 12: Possible species generated by metabolic cleavage of amidebonds. iL: cleavage forms a species with increased lipophilicity; DF:defluorination; nd: not detectable, since not radioactive.

FIG. 13: Left: Graphical analysis of overlapping peaks 1 (rhPSMA7.2) and2 (rhPSMA7.3); right: deconvolution and integration of peak profiles bySystat PeakFit Software); top: experimental data fitted, bottom:deconvoluted single peaks.

FIG. 14a : Quantification of relative changes (in % change of injectedracemic mixture) to evaluate the reproducibility of classicalintegration (by HPLC program) and deconvolution (by ‘PeakFit’) of peak 4(rhPSMA7.1). Both methods demonstrate similar performance for this peak.

FIG. 14b : Quantification of relative changes (in % change of injectedracemic mixture) to evaluate the reproducibility of classicalintegration (by HPLC program) and deconvolution (by ‘PeakFit’) of peak 3(rhPSMA7.4). Both methods demonstrate similar performance for this peak.

FIG. 15: Percentage change of each rhPSAM7.1-7.4 isomer in blood, liver,kidney, tumor and urine with respect to its proportion the injectedsolution ([¹⁸F][^(nat)Ga]rhPSMA7-rac. Data expressed as mean values±SD(n=4; see also FIG. 16).

FIG. 16: Percentage change of each rhPSAM7.1-7.4 isomer for each sampleand experiment) in blood, liver, kidney, tumor and urine with respect toits proportion in the injected solution ([¹⁸F][^(nat)Ga]rhPSMA7-rac).Analyses were carried out with Systat PeakFit.

FIG. 17: Left: TLC scanner profile of a TLC plate with liver sample(30.07.2018, overall cts: 142 cts). Due to their bad statistics andlimited validity dataset with cts<200 were removed. Right: Phophoimageof a TLC plate with liver sample: long tailing of the moving tracer.

FIG. 18: Left: TLC scanner profile of a TLC plate with a quality controlsample (01.08.2018, overall cts: 384). Right: Exemplay phophoimage of aTLC plate with Urine, kidney, liver, tumor, blood and QK sample.

FIG. 19: Radio-TLC of [F-18]rhPSMA7-rac (30.07.2018) as part of theQuality Control in the Department of Nuclear Medicine prior to clinicalapplication of the tracer. Note that a tailing of the tracer is evenobserved in the formulation buffer (and thus in the absence ofproteins).

FIG. 20: Quantification of free [F-18]Fluoride and ‘intact’[F-18]rhPSMA7-rac by radio-TLC of a urine sample (30.07.2018).

FIG. 21: Left: Radio-HPLC analysis of urine collected and pooled from 4normal mice injected with the respective [F-18]rhPSAM-7.x tracer. Right:Radio-HPLC analysis of ‘cold’ urine spiked with the respective[F-18]rhPSAM-7.x tracer for a period of 1 h (7.1., 7.2.), 0.5 h (7.3.)and 2 h (7.4.). HPLC-conditions: Solvent A: H20+0.1% TFA; Solvent B:MeCN+0.1% TFA; Gradient: 5% isocratic 0-3 min, 25-35% B 3-43 min, 95-95%B 43-48 min; flow: 1 mL/min, column: Nucleosil 100-5 C18, 125×4.6 mm

FIG. 22: Separation of radioactive species in urine by cartridgefixation and TLC. Top: Radio-HPLC analysis of urine 30 min p.i. of[F-18]rhPSMA7.3 in mice showing a small proportion at 1.6 min and intacttracer at ca. 34.5 min. Bottom (left): urine of mice, 30 min p.i. of[F-18]rhPSMA7.3, was diluted and subjected to STRATA-X cartridgefixation. The cartridge was washed and eluted with MeCN/water (60/40 v/v+1% TFA); only intact tracer was detected. Bottom (right): both thebreakthrough from the cartridge fixation (non-retained components) andthe fraction finally eluted MeCN/water from the cartridge were analysedby TLC (bottom, right). Whereas 96.1% [F-18]rhPSMA7.3 and only 3.9%[F-18]fluoride were found in the eluate of the cartridge, the reverseratio was found in the breakthrough of the cartridge (3.4%[F-18]rhPSMA7.3 and only 96.6% [F-18]fluoride)

FIG. 23: To fresh and nonradioactive urine of mice [F-18]rhPSMA7.3 wasadded, followed by 0.5 μmol cold F-19-fluoride; incubation for 2 h.Radioactivity was completely (98.5%) converted to a very hydrophilicfraction representing [F-18]fluoride (peak at 1.6 min). Note: peak at1.6 min was subsequently immobilized on a QMA cartridge and eluted withwith NaCl (1M) (=fluoride).

FIG. 24: Clinical biodistribution and uptake in tumor lesions of18F-rhPSMA-7 (left) and 18F-rhPSMA-7.3 (right) as demonstrated bySUVmax. Data are expressed as mean±SD.

FIG. 25: Clinical biodistribution and uptake in tumor lesions of¹⁸F-rhPSMA-7 (left) and ¹⁸F-rhPSMA-7.3 (right) as demonstrated bySUVmean. Data are expressed as mean±SD.

FIG. 26. Clinical biodistribution and uptake in tumor lesions of¹⁸F-rhPSMA-7 (left) and ¹⁸F-rhPSMA-7.3 (right) as demonstrated by theratio SUVmax to background. Data are expressed as mean±SD.

FIG. 27: Clinical biodistribution and uptake in tumor lesions of¹⁸F-rhPSMA-7 (left) and ¹⁸F-rhPSMA-7.3 (right) as demonstrated by theratio SUVmean to background. Data are expressed as mean±SD.

FIG. 28: Two clinical case examples of ¹⁸F-rhPSMA-7.3 PET-imaging.

The Examples illustrate the invention.

EXAMPLE 1 Material and Methods

The Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected aminoacid analogs were purchased from Bachem (Bubendorf, Switzerland) or IrisBiotech (Marktredwitz, Germany). The tritylchloride polystyrene (TCP)resin was obtained from PepChem (Tübingen, Germany). Chematech (Dijon,France) delivered the chelators DOTAGA-anhydride, (R)-DOTA-GA(tBu)₄ and(S)-DOTA-GA(tBu)₄. All necessary solvents and other organic reagentswere purchased from either, Alfa Aesar (Karlsruhe, Germany),Sigma-Aldrich (Munich, Germany) or VWR (Darmstadt, Germany). Solid phasesynthesis of the peptides was carried out by manual operation using anIntelli-Mixer syringe shaker (Neolab, Heidelberg, Germany). Analyticaland preparative reversed-phase high pressure chromatography (RP-HPLC)were performed using Shimadzu gradient systems (Shimadzu DeutschlandGmbH, Neufahrn, Germany), each equipped with a SPD-20A UV/Vis detector(220 nm, 254 nm). A Nucleosil 100 C18 (125×4.6 mm, 5 μm particle size)column (CS GmbH, Langerwehe, Germany) was used for analyticalmeasurements at a flow rate of 1 mL/min. Both specific gradients and thecorresponding retention times t_(R) are cited in the text. PreparativeHPLC purification was done with a Multospher 100 RP 18 (250×10 mm, 5 μmparticle size) column (CS GmbH, Langerwehe, Germany) at a constant flowrate of 5 mL/min. Analytical and preparative radio RP-HPLC was performedusing a Nucleosil 100 C18 (5 μm, 125×4.0 mm) column (CS GmbH,Langerwehe, Germany). Eluents for all HPLC operations were water(solvent A) and acetonitrile (solvent B), both containing 0.1%trifluoroacetic acid. Electrospray ionization-mass spectra forcharacterization of the substances were acquired on an expression^(L)CMS mass spectrometer (Advion Ltd., Harlow, UK). NMR spectra wererecorded on Bruker AVHD-300 or AVHD-400 spectrometers at 300 K. pHvalues were measured with a Seven Easy pH-meter (Mettler Toledo, Gießen,Germany).

Synthesis Protocols

1) Solid-Phase Peptide Synthesis Following the Fmoc-Strategy

TCP-Resin Loading (GP1)

Loading of the tritylchloride polystyrene (TCP) resin with aFmoc-protected amino acid (AA) was carried out by stirring a solution ofthe TCP-resin (1.95 mmol/g) and Fmoc-AA-OH (1.5 eq.) in anhydrous DCMwith DIPEA (4.5 eq.) at room temperature for 2 h. Remainingtritylchloride was capped by the addition of methanol (2 mL/g resin) for15 min. Subsequently the resin was filtered and washed with DCM (2×5mL/g resin), DMF (2×5 mL/g resin), methanol (5 mL/g resin) and dried invacuo. Final loading l of Fmoc-AA-OH was determined by the followingequation:

${l\left\lbrack \frac{mmol}{g} \right\rbrack} = \frac{\left( {m_{2} - m_{1}} \right) \times 1000}{\left( {M_{W} - M_{HCl}} \right)m_{2}}$

-   -   m₂=mass of loaded resin [g]    -   m₁=mass of unloaded resin [g]    -   M_(w)=molecular weight of AA [g/mol]    -   M_(HCl)=molecular weight of HCl [g/mol]

On-Resin Amide Bond Formation (GP2)

For conjugation of a building block to the resin bound peptide, amixture of TBTU and HOBT is used for pre-activation with DIPEA or2,4,6-trimethylpyridine as a base in DMF (10 mL/g resin) for 5 min. Theexact stoichiometry and reaction time for each conjugation step is givenin the synthesis protocol. After reaction, the resin was washed with DMF(6×5 mL/g resin).

On-Resin Fmoc-Deprotection (GP3)

The resin-bound Fmoc-peptide was treated with 20% piperidine in DMF(v/v, 8 mL/g resin) for 5 min and subsequently for 15 min. Afterwards,the resin was washed thoroughly with DMF (8×5 mL/g resin).

On-Resin Dde-Deprotection (GP4)

The Dde-protected peptide (1.0 eq.) was dissolved in a solution of 2%hydrazine monohydrate in DMF (v/v, 5 mL/g resin) and shaken for 20 min(GP4a). In the case of present Fmoc-groups, Dde-deprotection wasperformed by adding a solution of imidazole (0.92 g/g resin),hydroxylamine hydrochloride (1.26 g/g reisn) in NMP (5.0 mL) and DMF(1.0 mL) for 3 h at room temperature (GP4b). After deprotection theresin was washed with DMF (8×5 mL/g resin).

Peptide Cleavage from the Resin with Simultaneous Deprotection of AcidLabile Protecting Groups (GP 5)

The fully protected resin-bound peptide was dissolved in a mixture ofTFA/TIPS/water (v/v/v; 95/2.5/2.5) and shaken for 30 min. The solutionwas filtered off and the resin was treated in the same way for another30 min. Both filtrates were combined, stirred for additional 5 h andconcentrated under a stream of nitrogen. After dissolving the residue ina mixture of tert-butanol and water and subsequent lyophilisation thecrude peptide was obtained.

^(nat)Ga-Complexation (GP6)

For ^(nat)Ga-complexation, the peptide (1.0 eq.) was dissolved in a 3:1(v/v) mixture of tBuOH in H₂O and an aqueous solution of Ga(NO₃)₃ (3.5eq.) was added. After heating the resulting mixture for 30 min at 75° C.the peptide was purified by RP-HPLC.

2) Synthesis of the PSMA Binding Motif

Glu-urea-Glu ((tBuO)EuE(OtBu)2)

The tBu-protected Glu-urea-Glu binding motif (EuE) was synthesizedaccording to a previously published procedure (scheme 1) fortBu-protected Glu-urea-Lys (EuK)¹. ¹Wemeisen, M.; Simecek, J.;Schottelius, M.; Schwaiger, M.; Wester, H. J., Synthesis and preclinicalevaluation of DOTAGA-conjugated PSMA ligands for functional imaging andendoradiotherapy of prostate cancer. EJNMMI research 2014, 4 (1), 63.

Di-tert-butyl (1H-imidazole-1-carbonyl)-L-glutamate (i)

A solution of DCM containing 2.0 g (7.71 mmol, 1.0 eq.)I-di-tert-butyl-L-glutamate.HCl was cooled on ice for 30 min andafterwards treated with 2.69 mL TEA (19.28 mmol, 2.5 eq.) and 3.3 mg(0.3 mmol, 0.04 eq.) DMAP. After additional stirring for 5 min, 1.38 g(8.84 mmol, 1.1 eq.) of 1,1′-carbonyldiimidazole (CDI) dissolved in DCMwere slowly added over a period of 30 min. The reaction mixture wasfurther stirred overnight and enabled to warm to RT. The reaction wasstopped using 8 mL saturated NaHCO₃ with concomitant washing steps ofwater (2×) and brine (2×) and dried over Na₂SO_(4.) The remainingsolvent was removed in vacuo and the crude product (S)-Di-tert-butyl2-(1H-imidazole-1-carboxamido)pentanedioate (i) was used without furtherpurification.

5-benzyl 1-(tert-butyl)(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate(ii)

2.72 g (7.71 mmol, 1.0 eq.) of the crude product(S)-Di-tert-butyl-2-(1H-imidazole-1-carboxamido) pentanedioate (i) weredissolved in 1,2-dichloroethane (DCE) and cooled on ice for 30 min. Tothis solution were added 2.15 mL (15.42 mmol, 2.0 eq.) TFA and 2.54 g(7.71 mmol, 1.0 eq.) H-L-Glu(OBzl)-OtBu.HCl and the solution was stirredovernight at 40° C. The remaining solvent was evaporated and the crudeproduct purified using silica gel flash-chromatography with an eluentmixture containing ethyl acetate/hexane/TFA (500:500:0.8; v/v/v). Afterremoval of the solvent,5-benzyl-1-(tert-butyl)-WS)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate(ii) was obtained as a colorless oil.

(tBuO)EuE(OtBu)₂ (iii)

To synthesize (tBuO)EuE(OtBu)₂, 3.17 g (5.47 mmol, 1.0 eq.) of5-benzyl-1-(tert-butyl)-(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate(ii) were dissolved in 75 mL EtOH and 0.34 g (0.57 mmol, 0.1 eq.)palladium on activated charcoal (10%) were given to this solution. Theflask containing the reaction mixture was initially purged with H₂ andthe solution was stirred over night at room temperature under lightH₂-pressure (balloon). The crude product was purified through celite andthe solvent evaporated in vacuo. The product (iii) was obtained as ahygroscopic solid (84%). HPLC

(10% to 90% B in 15 min): t_(R)=11.3 min. Calculated monoisotopic mass(C₂₃H₄₉N₂O₉): 488.3; found: m/z=489.4 [M+H]⁺, 516.4 [M+Na]⁺.

3) Synthesis of the Silicon-Fluoride Acceptor

4-(Di-tert-butylfluorosilyl)benzoic acid (SiFA-BA)

SiFA-BA was synthesized according to a previously published procedure(scheme 2)². All reactions were carried out in dried reaction vesselsunder argon using a vacuum gas manifold. ²Iovkova, L.; Wangler, B.;Schirrmacher, E.; Schirrmacher, R.; Quandt, G.; Boening, G.; Schurmann,M.; Jurkschat, K., para-Functionalized aryl-di-tert-butylfluorosilanesas potential labeling synthons for (18)F radiopharmaceuticals. Chemistry(Weinheim an der Bergstrasse, Germany) 2009, 15 (9), 2140-7.

((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane (i)

To a stirred solution of 4-bromobenzylalcohol (4.68 g, 25.0 mmol, 1.0eq.) in anhydrous DMF (70 mL) imidazole (2.04 g, 30.0 mmol, 1.2 eq.) andTBDMSCI (4.52 g, 30.0 mmol, 1.2 eq.) were added and the resultingmixture was stirred at room temperature for 16 h. The mixture was thenpoured into ice-cold H₂O (250 mL) and extracted with Et₂O (5×50 mL). Thecombined organic fractions were washed with sat. aq. NaHCO₃ (2×100 mL)and brine (100 mL), dried, filtered and concentrated in vacuo to givethe crude product which was purified by flash column chromatography(silica, 5% EtOAc/petrol) to give i as a colourless oil (7.18 g, 95%).¹H NMR (400 MHz, CDCl3): δ [ppm]=0.10 (6H, s, SiMe₂t-Bu), 0.95 (9H, s,SiMe₂tBu), 4.69 (2H, s, CH₂OSi), 7.21 (2H, d), 7.46 (2H, d). HPLC (50 to100% B in 15 min): t_(R)=15 min.

Di-tert-butyl{4-[(tert-butyldimethylsilyloxy)methyl]phenyl}fluorosilane(ii)

At −78° C. under magnetic stirring, a solution of tBuLi in pentane (7.29mL, 1.7 mol/L, 12.4 mmol 2.4 eq.) was added to a solution of((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane (i) (1.56 g, 5.18 mmol,1.0 eq.) in dry THF (15 mL). After the reaction mixture had been stirredfor 30 min at −78° C., the suspension obtained was added dropwise over aperiod of 30 min to a cooled (−78° C.) solution ofdi-tert-butyldifluorosilane (1.12 g, 6.23 mmol, 1.2 eq.) in dry THF (10mL). The reaction mixture was allowed to warm to room temperature over aperiod of 12 h and then hydrolyzed with saturated aqueous NaCl solution(100 mL). The organic layer was separated and the aqueous layer wasextracted with diethyl ether (3×50 mL). The combined organic layers weredried over magnesium sulfate and filtered. The filtrate was concentratedin vacuo to afford ii as a yellowish oil (1.88 g, 95%). It was used forsubsequent reactions without further purification. NMR spectra were inaccordance with the data reported in the literature^([2]). HPLC (50 to100% B in 20 min): t_(R)=19 min.

4-(Di-tert-butylfluorosilanyl)benzyl alcohol (iii)

A catalytic amount of concentrated aqueous HCl (0.5 mL) was added to asuspension of ii (1.88 g, 4.92 mmol, 1.0 eq.) in methanol (50 mL). Thereaction mixture was stirred for 18 h at room temperature and then thesolvent and the volatiles were removed under reduced pressure. Theresidue was redissolved in diethyl ether (40 mL) and the solution waswashed with saturated aqueous NaHCO₃ solution. The aqueous layer wasextracted with diethyl ether (3×50 mL). The combined organic layers weredried over magnesium sulfate and filtered. The filtrate was concentratedin vacuo to afford iii as a yellowish oil (1.29 g, 98%) that solidified.The product was used without further purification. NMR spectra were inaccordance with the data reported in the literature^([2]). HPLC (50 to100% B in 15 min): t_(R)=8.2 min.

4-(Di-tert-butylfluorosilyl)benzaldehyde (iv)

A solution of the alcohol iii (1.37 g, 5.10 mmol, 1.0 eq.) in drydichloromethane (20 mL) was added dropwise to a stirred ice-cooledsuspension of pyridinium chlorochromate (2.75 g, 12.8 mmol, 2.5 eq.) indry dichloromethane (60 mL). After the reaction mixture had been stirredfor 30 min at 0° C. and for 2.5 h at room temperature, anhydrous diethylether (40 mL) was added and the supernatant solution was decanted fromthe black gum-like material. The insoluble material was washedthoroughly with diethyl ether and the combined organic phases werepassed through a short pad of silica gel (10 cm per g crude product) forfiltration. The solvents were removed in vacuo to yield aldehyde iv as ayellowish oil (1.31 g, 96%). NMR spectra were in accordance with thedata reported in the literature^([2]). HPLC (50 to 100% B in 15 min):t_(R)=10.5 min.

4-(Di-tert-butylfluorosilyl)benzoic acid (v)

At room temperature, 1 M aqueous KMnO₄ (30 mL) was added to a mixture ofiv (1.31 g, 4.92 mmol, 1.0 eq.), tert-butanol (30 mL), dichloromethane(3.3 mL), and 1.25 M NaH₂PO₄.H₂O buffer (20 mL) at pH 4.0-4.5. After themixture had been stirred for 25 min, it was cooled to 5° C., whereuponexcess KMnO₄ (0.78 g, 4.92 mmol, 1.0 eq.) was added. The reaction wasthen quenched by the addition of saturated aqueous Na₂SO₃ solution (50mL). Upon addition of 2 M aqueous HCl, all of the MnO₂ dissolved. Theresulting solution was extracted with diethyl ether (3×100 mL). Thecombined organic layers were washed with saturated aqueous NaHCO₃solution, dried over MgSO₄, filtered, and concentrated under reducedpressure to provide a white solid, which was purified byrecrystallization from Et₂O/n-hexane (1:3, for 12 h) to give v (0.84 g,60%). NMR spectra were in accordance with the data reported in theliterature^([2]). HPLC (50 to 100% B in 15 min): t_(R)=8.5 min.

4) Synthesis of rhPSMA-7.1-7.4

The first synthetic steps for preparation of the four different isomersof rhPSMA-7 are identical and carried out together, applying thestandard Fmoc-SPPS protocol described above, starting from resin boundFmoc-D-Orn(Dde)-OH. After cleavage of the Fmoc group with 20% piperidinein DMF (GP3), (tBuO)EuE(OtBu)₂ (2.0 eq.) was conjugated with HOAt (2.0eq.), TBTU (2.0 eq.) and DIPEA (6.0 eq.) in DMF for 4.5 h. Aftercleavage of the Dde-group with a mixture of 2% hydrazine in DMF (GP4a),a solution of succinic anhydride (7.0 eq.) and DIPEA (7.0 eq.) in DMFwas added and left to react for 2.5 h. Conjugation ofFmoc-D-Lys(OtBu).HCl (2.0 eq.) was achieved by adding a mixture of HOAt(2.0 eq.), TBTU (2.0 eq.) and DIPEA (6.0 eq.) in DMF to the resin. Afterpre-activation for 5 min, Fmoc-D-Lys(OtBu).HCl (2.0 eq.) dissolved inDMF was added and left to react for 2.5 h (GP2). Subsequent cleavage ofthe Fmoc-group was performed, by adding a mixture of 20% piperidine inDMF (GP3). Finally, the resin was split in order to synthesizerhPSMA-7.1-7.4 (scheme 3).

rhPSMA-7.1 (D-Dap-(R)-DOTA-GA):

Fmoc-D-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF andadded to the resin-bound peptide for 2.5 h. Following orthogonalDde-deprotection was done using imidazole and hydroxylaminehydrochloride dissolved in a mixture of NMP and DMF for 3 h. SiFA-BA(1.5 eq.) was reacted with the free amine of the side chain with HOAt(1.5 eq.), TBTU (1.5 eq.) and DIPEA (4.5 eq.), as activation reagents inDMF for 2 h. After Fmoc-deprotection with piperidine (GP3),(R)-DOTA-GA(tBu)₄ (2.0 eq.) was conjugated with HOAT (2.0 eq.), TBTU(2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h.Cleavage from the resin with simultaneous deprotection of acid labileprotecting groups was performed in TFA, according to GP5.^(nat)Ga-complexation of the peptide was carried out, as described inGP6.

rhPSMA-7.2 (L-Dap-(R)-DOTA-GA):

Fmoc-L-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for2.5 h. Following orthogonal Dde-deprotection, conjugation of SiFA-BA andFmoc-cleavage was carried out as described for rhPSMA-7.1.(R)-DOTA-GA(tBu)₄ (2.0 eq.) was conjugated with HOAT (2.0 eq.), TBTU(2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h.Cleavage from the resin with simultaneous deprotection of acid labileprotecting groups was performed in TFA according to GP5.^(nat)Ga-complexation of the peptide was carried out, as described inGP6.

rhPSMA-7.3 (D-Dap-(S)-DOTA-GA):

Fmoc-D-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for2.5 h. Following orthogonal Dde-deprotection, conjugation of SiFA-BA andFmoc-cleavage was carried out as described for rhPSMA-7.1.(S)-DOTA-GA(tBu)₄ (2.0 eq.) was conjugated with HOAT (2.0 eq.), TBTU(2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h.Cleavage from the resin with simultaneous deprotection of acid labileprotecting groups was performed in TFA according to GP5.^(nat)Ga-complexation of the peptide was carried out, as described inGP6.

rhPSMA-7.4 (L-Dap-(S)-DOTA-GA):

Fmoc-L-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for2.5 h. Following orthogonal Dde-deprotection, conjugation of SiFA-BA andFmoc-cleavage was carried out as described for rhPSMA-7.1.(S)-DOTA-GA(tBu)₄ (2.0 eq.) was conjugated with HOAT (2.0 eq.), TBTU(2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h.Cleavage from the resin with simultaneous deprotection of acid labileprotecting groups was performed in TFA according to GP5.^(nat)Ga-complexation of the peptide was carried out, as described inGP6.

rhPSMA-7.1:

HPLC (10 to 70% B in 15 min): t_(R)=10.5 min.

HPLC (25 to 35%B in 40 min): t_(R)=31.4 min.

rhPSMA-7.2:

HPLC (10 to 70% B in 15 min): t_(R)=10.4 min.

HPLC (25 to 35%B in 40 min): t_(R)=27.9 min.

rhPSMA-7.3:

HPLC (10 to 70% B in 15 min): t_(R)=10.4 min.

HPLC (25 to 35%B in 40 min): t_(R)=28.1 min.

rhPSMA-7.4:

HPLC (10 to 70% B in 15 min): t_(R)=10.5 min.

HPLC (25 to 35%B in 40 min): t_(R)=29.1 min.

rhPSMA-7.1-7.4:

Calculated monoisotopic mass (C63H96FGaN12O25Si): 1536.6 found:m/z=1539.4 [M+H]+, 770.3 [M+2H]2⁺.

5) ¹⁸F-Labeling

For ¹⁸F-labeling a previously published procedure³ was applied, whichwas slightly modified. Briefly, aqueous ¹⁸F was passed through a SAXcartridge (Sep-Pak Accell Plus QMA Carbonate light), which waspreconditioned with 10 mL of water. After drying with 10 mL of air,water was removed, by rinsing the cartridge with 10 mL of anhydrousacetonitrile followed by 20 mL of air. ¹⁸F was eluted with 100 μmol of[K⁺⊂2.2.2]OH⁻ dissolved in 500 μL of anhydrous acetonitrile. Beforelabelling, 30 μmol of oxalic acid in anhydrous acetonitrile (1 M, 30 μL)were added. This mixture was used as a whole or aliquot for fluorinationof 10-25 nmol of PSMA-SiFA (1 mM in anhydrous DMSO). The resultingreaction mixture was incubated for 5 minutes at room temperature. Forpurification of the tracer, a Sep-Pak C18 light cartridge,preconditioned with 10 mL EtOH, followed by 10 mL of H₂O was used. Thelabeling mixture was diluted with 9 mL PBS (pH 3) and passed through thecartridge followed by 10 mL of H₂O. The peptide was eluted with 500 μLof a 4:1 mixture (v/v) of EtOH in water. Radiochemical purity of thelabelled compound was determined by radio RP-HPLC and radio-TLC (Silicagel 60 RP-18 F₂₅₄s, mobile phase: 3:2 mixture (v/v) of MeCN in H₂Osupplemented with 10% of 2 M aqueous NaOAc and 1% of TFA). ³Wangler, C.;Niedermoser, S.; Chin, J.; Orchowski, K.; Schirrmacher, E.; Jurkschat,K.; Iovkova-Berends, L.; Kostikov, A. P.; Schirrmacher, R.; Wangler, B.,One-step (18)F-labeling of peptides for positron emission tomographyimaging using the SiFA methodology. Nat Protoc 2012, 7 (11), 1946-55.

6)¹²⁵I-Labeling

The reference ligand for in vitro studies ([¹²⁵I]I-BA)KuE was preparedaccording to a previously published procedure¹. Briefly, 0.1 mg of thestannylated precursor (SnBu₃-BA)(OtBu)KuE(OtBu)₂ was dissolved in asolution containing 20 μL peracetic acid, 5.0 μL (21 MBq) [¹²⁵I]NaI (74TBq/mmol, 3.1 GBq/mL, 40 mM NaOH, Hartmann Analytic, Braunschweig,Germany), 20 μL MeCN and 10 μL acetic acid. The reaction solution wasincubated for 10 min at RT, loaded on a cartridge and rinsed with 10 mLwater (C18 Sep Pak Plus cartridge, preconditioned with 10 mL MeOH and 10mL water). After elution with 2.0 mL of a 1:1 mix (v/v) of EtOH/MeCN,the radioactive solution was evaporated to dryness under a gentlenitrogen stream and treated with 200 μL TFA for 30 min with subsequentevaporation of TFA. The crude product of ([¹²⁵I]I-BA)KuE was purified byRP-HPLC (20% to 40% B in 20 min): t_(R)=13.0 min.

In Vitro Experiments

1) Determination of IC₅₀

The PSMA-posivite LNCaP cells were grown in Dublecco modified Eaglemedium/Nutrition Mixture F-12 with Glutamax-I (1:1) (Invitrigon),supplemented with 10% fetal calf serum and maintained at 37° C. in ahumidified 5% CO₂ atmosphere. For determination of the PSMA affinity(IC₅₀), cells were harvested 24±2 hours before the experiment and seededin 24-well plates (1.5×10⁵ cells in 1 mL/well). After removal of theculture medium, the cells were treated once with 500 μL of HBSS (Hank'sbalanced salt solution, Biochrom, Berlin, Germany, with addition of 1%bovine serum albumin (BSA)) and left 15 min on ice for equilibration in200 μL HBSS (1% BSA). Next, 25 μL per well of solutions, containingeither HBSS (1% BSA, control) or the respective ligand in increasingconcentration (10⁻¹⁰-10⁻⁴ M in HBSS, were added with subsequent additionof 25 μL of ([¹²⁵I]I-BA)KuE (2.0 nM) in HBSS (1% BSA). All experimentswere performed at least three times for each concentration. After 60 minincubation on ice, the experiment was terminated by removal of themedium and consecutive rinsing with 200 μL of HBSS. The media of bothsteps were combined in one fraction and represent the amount of freeradioligand. Afterwards, the cells were lysed with 250 μL of 1 M NaOHand united with the 200 μL HBSS of the following wash step.Quantification of bound and free radioligand was accomplished in aγ-counter.

2) Internalization

For internalization studies, LNCaP cells were harvested 24±2 hoursbefore the experiment and seeded in 24-well plates (1.25×10⁵ cells in 1mL/well). Subsequent to the removal of the culture medium, the cellswere washed once with 500 μL DMEM-F12 (5% BSA) and left to equilibratefor at least 15 min at 37° C. in 200 μL DMEM-F12 (5% BSA). Each well wastreated with either 25 μL of either DMEM-F12 (5% BSA) or a 100 μM PMPAsolution for blockade. Next, 25 μL of the ⁶⁸Ga/¹⁸F-labeled PSMAinhibitor (5.0 nM) was added and the cells incubated at 37° C. for 60min. The experiment was terminated by placing the 24-well plate on icefor 3 min and consecutive removal of the medium. Each well was rinsedwith 250 μL HBSS and the fractions from these first two steps combined,representing the amount of free radioligand. Removal of surface boundactivity was accomplished by incubation of the cells with 250 μL ofice-cold PMPA (10 μM in PBS) solution for 5 min and rinsed again withanother 250 μL of ice-cold PBS. The internalized activity was determinedby incubation of the cells in 250 μL 1 M NaOH and the combination withthe fraction of a subsequent wash step with 250 μL 1.0 M NaOH. Eachexperiment (control and bloackde) was performed in triplicate. Free,surface bound and internalized activity was quantified in a γ-counter.All internalization studies were accompanied by reference studies using([¹²⁵I]I-BA)KuE (c=0.2 nM), which were performed analogously. Data werecorrected for non-specific internalization and normalized to thespecific-internalization observed for the radioiodinated referencecompound.

3) Octanol-Water Partition Coefficient

Approximately 1 MBq of the labeled tracer was dissolved in 1 mL of a 1:1mixture (by volumes) of phosphate buffered saline (PBS, pH 7.4) andn-octanol in an Eppendorf tube. After vigorous mixing of the suspensionfor 3 minutes at room temperature, the vial was centrifuged at 15000 gfor 3 minutes (Biofuge 15, Heraus Sepatech, Osterode, Germany) and 100μL aliquots of both layers were measured in a gamma counter. Theexperiment was repeated at least six times.

4) HSA Binding

For the determination of HSA binding, a Chiralpak HSA column (50×3 mm, 5μm, H13H-2433) was used at a constant flow rate of 0.5 mL/min. Themobile phase (A: NH₄OAc, 50 mM in water, pH 7 and B: isopropanol) wasfreshly prepared for each experiment and only used for one day. Thecolumn was kept at room temperature and each run was stopped afterdetection of the signal to reduce the acquisition time. All substanceswere dissolved in a 0.5 mg/ml concentration in 50% 2-propanol and 50% 50mM pH 6.9 ammonium acetate buffer. The chosen reference substancesdisplay a range of HSA binding from 13% to 99% since a broad variety ofalbumin binding regarding the peptides was assumed. All nine referencesubstances were injected consecutively to establish a non-linearregression with Origin Pro 2016G.

TABLE 1 Reference substances⁴ used for the calibration of theHSA-column. Reference t_(R) Log t_(R) Lit. HSA % Log K HSAp-benzylalcohol 2.40 0.38 13.15 −0.82 Aniline 2.72 0.43 14.06 −0.79Phenol 3.28 0.52 20.69 −0.59 Benzoic Acid 4.08 0.61 34.27 −0.29Carbamazepine 4.15 0.62 75.00 0.46 p-nitrophenol 5.62 0.75 77.65 0.52Estradiol 8.15 0.91 94.81 1.19 Probenecid 8.84 0.95 95.00 1.20Glibenclamide 29.18 1.47 99.00 1.69 The retention time is shownexemplary for a conducted experiment; t_(R) retention time; Lit. HSAliterature value of human serum albumin binding in [%]; Log K HASlogarithmic K of human serum albumin binding. ⁴Yamazaki, K.; Kanaoka,M., Computational prediction of the plasma protein-binding percent ofdiverse pharmaceutical compounds. Journal of pharmaceutical sciences2004, 93 (6), 1480-94.

In Vivo Experiments

All animal experiments were conducted in accordance with general animalwelfare regulations in Germany and the institutional guidelines for thecare and use of animals. To establish tumor xenografts, LNCaP cells (10⁷cells/200 μL) were suspended in a 1:1 mixture (v/v) of Dulbecco modifiedEagle medium/Nutrition Mixture F-12 with Glutamax-I (1:1) and Matrigel(BD Biosciences, Germany), and inoculated subcutaneously onto the rightshoulder of 6-8 weeks old CB17-SCID mice (Charles River, Sulzfeld,Germany). Mice were used for when tumors had grown to a diameter of 5-8mm (3-4 weeks after inoculation).

1) Biodistribution

Approximately 1-2 MBq (<0.2 nmol) of the ¹⁸F-labeled PSMA inhibitor wasinjected into the tail vein of LNCaP tumor-bearing male CB-17 SCID miceand sacrificed after 1 h post injection (n=4-5). Selected organs wereremoved, weighted and measured in a γ-counter

2) Metabolism Studies

a) Analytical Set-Up

Analytical reversed-phase high pressure chromatography (RP-HPLC) wereperformed using Shimadzu gradient systems (Shimadzu Deutschland GmbH,Neufahrn, Germany), equipped with a SPD-20A UV/Vis detector (220 nm, 254nm). A Multospher 100 RP18 (125×4.6 mm, 5 μm particle size) column (CSGmbH, Langerwehe, Germany) was used for analytical measurements at aflow rate of 1 mL/min. Eluents for all HPLC operations were water(solvent A) and acetonitrile (solvent B), both containing 0.1%trifluoroacetic acid. Radioactivity was detected through connection ofthe outlet of the UV-photometer to a HERM LB 500 detector (BertholdTechnologies GmbH, Bad Wildbad, Germany). The gradient for all HPLCoperations was: 5% B isocratic 0-3 min, 25-35% B 3-43 min, 95-95% B43-48 min.

For radio-thin-layer chromatography, aluminum sheets coated with silicagel 60 RP-18 F₂₅₄s were used with a mobile phase consisting of a 3:2mixture (v/v) of MeCN in H₂O supplemented with 10% of 2 M aqueous NaOAcand 1% of TFA. Analysis was performed using either a Scan-RAM radio-TLCdetector (LabLogic Systems Ltd., Sheffield, United Kingdom) or a CR 35BIO phosphorimager (Duerr Medical GmbH, Bietigheim-Bissingen, Germany).

b) Determination of Metabolic Stability of rhPSMA-7.1-7.4

For in vivo μmetabolism studies, 8-12 MBq (<0.6 nmol) of the respective¹⁸F-labeled ligand (rhPSMA-7.1-7.4) was injected into the tail vein offemale healthy CB17-SCID mice (n=4). Mice were left under anesthesia for30 min and the urine was collected using a bladder catheter. Urinesamples were pooled and centrifuged for 5 min at 9000 rpm to removesuspended solids. The supernatant was directly used for radio-HPLCanalysis with the above mentioned conditions. In order to demonstratethat isotopic exchange of ¹⁹F with peptide-bound ¹⁸F is taking place inurine, each compound was incubated for certain time intervals with urinesamples of female healthy CB-17-SCID mice, which where analysed byradio-HPLC and/or radio-TLC. Additionally, this experiment was carriedout with the addition of excess Na¹⁹F (0.5 μmol) and incubation for 2 hwith ¹⁸F-labeled rhPSMA-7.3.

c) Determination of In Vivo Distribution of rhPSMA-7.1-7.4

In order to quantify the relative uptake of each isomer(rhPSMA-7.1-7.4), a tumor-bearing male CB-17-SCID mouse was injectedwith the racemic mixture of rhPSMA-7 (180-280 MBq, S_(A)=247-349GBq/μmol, produced at the Klinikum rechts der Isar in a fully automatedprocedure). The animal was left under anesthesia for 30 min andsacrificed. Urine, blood, liver, kidneys and tumor were collected andprocessed to the hereafter described procedures. The urine sample wascentrifuged for 5 min at 9000 rpm to yield a clear solution and directlysubjected to radio-HPLC analysis. Blood was diluted to 1 mL with H₂O andcentrifuged twice at 13000 g for 5 min. The supernatant was collectedand loaded on a Strata X cartridge (33 μm Polymeric Reversed Phase 500mg, pre-conditioned with 5 mL MeOH, followed by 5 mL H₂O). After washingwith 5 mL H₂O, the cartridge was eluted with a 6:4 mixture (v/v) of MeCNin H₂O, supplemented with 1% TFA. The eluate was diluted with water andanalysed by radio-HPLC. Tumour, kidneys and liver were homogenised usingeither a Potter-Elvehjem tissue grinder (Kontes Glass Co, Vineland, USA)or a MM-400 ball mill (Retsch GmbH, Haan, Germany).

I) Potter-Elvehjem Tissue Grinder

Tumour and kidneys were separately homogenised in the tissue homogeniserwith 1 mL of extraction buffer (850 μL 1 M HEPES pH7.4, 100 μL20 mM PMPAand 100 μL 1M NaCl) for 30 min. The resulting homogenate was collectedand centrifuged at 13000 g for 5 min. Subsequently the supernatant wascollected, centrifuged again (13000 g, 5 min) and loaded on a Strata Xcartridge (33 μm Polymeric Reversed Phase 500 mg, pre-conditioned with 5mL MeOH, followed by 5 mL H₂O). After washing with 5 mL H₂O, thecartridge was eluted with a 6:4 mixture (v/v) of MeCN in H₂O,supplemented with 1% TFA. The eluate of each organ was diluted withwater and analysed by radio-HPLC.

II) MM-400 Ball Mill

The organs (tumour, kidney, liver) were separately homogenised in a 2 mLtube together with 3 grinding balls (3 mm diameter) and 1 mL ofextraction buffer (850 μL 1 M HEPES pH7.4, 100 μL 20 mM PMPA and 100 μL1M NaCl) for 10 min at 30 Hz. The homogenate was centrifuged at 13000 gfor 5 min and the supernatant was collected. Subsequently, the pelletwas suspended in 1 mL of extraction buffer and homogenized again withthe ball mill for 10 min at 30 Hz. After centrifugation (13000 g, 5min), both supernatants were combined and loaded on a Strata X cartridge(33 μm Polymeric Reversed Phase 500 mg, pre-conditioned with 5 mL MeOH,followed by 5 mL H₂O). After washing with 5 mL H₂O, the cartridge waseluted with a 6:4 mixture (v/v) of MeCN in H₂O, supplemented with 1%TFA. The eluate of each organ was diluted with water and analysed byradio-HPLC. In order to demonstrate that the breakthrough duringcartridge loading, is not a result of unbound F-18, the supernatant wasalso examined by radio-TLC after centrifugation. Finally the ratios ofthe individual isomers were determined from the HPLC profiles of theextracted samples and compared to the ratios of the isomers from thequality control of the racemic mixture of rhPSMA-7. The decay correctedextraction- and cartridge loading-efficiency, as well as the overallextracted activity of the examined samples are given in table 2. Thecartridge elution-efficiency was >99% for all experiments.

EXAMPLE 2 Results

Chromatographic Peak Assignment

The chromatographic peak assignment was carried out by comparison of theUV profiles of

a) the rhPSMA7-rac micture with

b) the rhPSMA7-rac micture coninjected with each enantiopure rhPSMA7compound.

The following names are used for the different isomers:

rhPSMA-rac: [¹⁹F][^(nat)Ga]D/L-Dap-R/S-DOTAGA-rhPSMA7

rhPSMA-7-1: [¹⁹F][^(nat)Ga]D-Dap-R-DOTAGA-rhPSMA7

rhPSMA-7-2: [¹⁹F][^(nat)Ga]L-Dap-R-DOTAGA-rhPSMA7

rhPSMA-7-3: [¹⁹F][^(nat)Ga]D-Dap-S-DOTAGA-rhPSMA7

rhPSMA-7-4: [¹⁹F][^(nat)Ga]L-Dap-S-DOTAGA-rhPSMA7

TABLE 2 Assignment of the different isomers, names, typical retentiontimes (HPLC conditions are given in FIG. 2a-4b, and percentage of eachisomer on a typical rhPSAM7-rac mixture. The exact amount can vary foreach isomer. typical percentage of ligand Name t_(R) [min] whole mixture[¹⁹F][^(nat)Ga]D-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-1 31.6 21[¹⁹F][^(nat)Ga]L-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-2 28.3 22[¹⁹F][^(nat)Ga]D-Dap-S-DOTAGA-rhPSMA7 rhPSMA-7-3 28.9 37[¹⁹F][^(nat)Ga]L-Dap-S-DOTAGA-rhPSMA7 rhPSMA-7-4 30.1 20

Binding Affinities

The first set of values (rhPSMA-7.1 and rhPSMA-7.2; FIG. 6a ) weredetermined by using for the dilution series a solution directly obtainedafter ^(nat)Ga-complexation of the respective ligand. In the second dataset (FIG. 6b ), the complexed ligands were purified by RP-HPLC in orderto separate uncomplexed ^(nat)Ga-salts. Since there were no significantdifferences observed, both series were merged and used for thecalculation of the mean values (±SD).

TABLE 3 Depiction of the individual IC₅₀ [nM] measurements (as shown inFIG. 6a and 6b). Conditions as described in the legend to FIG. 6a. NorhPSMA7.1 rhPSMA7.2 rhPSMA7.3 rhPSMA7.4 1 8.74 3.17 nd nd 2 6.91 2.97 ndnd 3 7.27 3.36 nd nd 4 5.04 2.64 3.17 3.4 5

2.76 2.91 3.56 6 7.11 3.94 5.35 2.79 7 8.31 5.8 5.74 4.57 8 4.97 4.314.59 3.78 9 6.44 4.32 4.45 nd Mean 6.85 3.70 4.37 3.62 SD 1.36 1.01 1.140.65 *Value no 5 of the rhPSMA7.1 series was deleted (statisticaloutlier).

TABLE 4 Binding affinities (IC₅₀ [nM]) of other selected PSMA inhibitors(*). No Inhibitor IC₅₀ [nM] 1 (I-BA)KuE 7.1 ± 2.4 nM 2 DCFPyL 12.3 ± 1.2nM  3 DKFZ1007 4.2 ± 0.5 nM * carried out in our lab using the identicalbinding assay (Robu et al. EJNMMI Research 2018; 8:30).

Internalization Studies

TABLE 5 Depiction of the individual internalization rates [% of[¹²⁵I]IB-KuE]. rhPSMA7.1 rhPSMA7.2 rhPSMA7.3 rhPSMA7.4 1 61.8 188.7156.6 209.6 1 70.6 182.7 156.0 202.6 1 68.0 169.5 171.7 209.8 1 67.9205.3 — — 1 71.5 212.6 — — 1 77.5 192.3 — — Mean 69.55 191.83 161.41207.33 SD 5.19 15.54 8.88 4.06

TABLE 6 Internalization values [% of [¹²⁵I][IB-KuE] of other selectedPSMA inhibitors (*). No Inhibitor internalization [%] 1 PSMA-1007 118 ±4 2 DCFPyL 118 ± 5 * carried out in our lab using the identical bindingassay (Robu et al. EJNMMI Research 2018; 8:30).

Lipophilicities (Octanol-Water Partition Coefficient)

Determianation of the logP values was carried out in phosphate bufferedsaline (PBS, pH 7.4) and n-octanol (=logP_(oct/PBS)).

TABLE 7 Individual log P measurements for rhPSMA7-isomers 7.1-7.4isomers, determined in octanol/PBS_(7.4) mixtures. rhPSMA7.1 rhPSMA7.2rhPSMA7.3 rhPSMA7.4 rhPSMA7-rac 1 −2.79 −3.00 −3.03 −3.09 −3.23 2 −2.82−3.01 −3.08 −3.12 −3.15 3 −2.80 −3.00 −3.07 −3.06 −3.17 4 −2.84 −3.00−3.02 −3.07 −3.10 5 −2.88 −3.03 −3.06 −3.10 −3.17 6 −2.90 −2.96 −3.07−3.04 −3.24 7 −2.85 −2.94 −3.06 −2.99 −3.80 8 −2.86 −2.89 −2.86 −2.97−3.60 9 −3.14 −3.02 −3.39 −3.37 −3.87 10 −3.29 −3.02 −3.33 −3.43 −3.6111 −3.26 −3.06 −3.34 −3.29 −3.76 12 −3.02 −3.02 −3.34 −3.48 −3.65 13−3.15 −2.99 −3.20 −3.52 −3.67 14 −3.57 −3.02 −3.39 −3.50 — 15 −3.40−3.06 −3.40 −3.44 — 16 −3.32 −3.14 −3.41 −3.41 — 17 −3.64 −3.40 −3.48−3.56 — 18 −3.92 −3.50 −3.49 −3.61 — 19 — −3.45 −3.32 −3.58 — 20 — −3.45−3.45 −3.54 — 21 — −3.53 −3.53 −3.42 — 22 — −3.48 −3.43 −3.57 — 23 — —−3.56 −3.67 — Mean −3.14 −3.13 −3.26 −3.33 −3.46 SD 0.34 0.22 0.19 0.220.29

TABLE 8 log P values of PSMA-1007, DCFPYL, rhPSMA7-rac and rhPSAM7.1-7.4isomers; (n = 6), octanol/PBS_(7.4). Inhibitor log P PSMA-1007 −1.6DCFPyL −3.4 ^(nat)Ga-¹⁸F-rhPSMA7-rac, −3.46 ± 0.29⁶⁸Ga-^(nat)F-rhPSMA7-rac ^(nat)Ga-¹⁸F-rhPSMA7.1 −3.14 ± 0.34^(nat)Ga-¹⁸F-rhPSMA7.2 −3.13 ± 0.22 ^(nat)Ga-¹⁸F-rhPSMA7.3 −3.26 ± 0-19^(nat)Ga-¹⁸F-rhPSMA7.4 −3.33 ± 0.22

Binding of PSMA Inhibitors to Human Plasma Protein

TABLE 9 HSA binding of of PSMA-1007, DCFPYL, rhPSMA7-rac andrhPSAM7.1-7.4 isomers; (n = 6). Determined on a Chiralpak HSA column (50× 3 mm, 5 μm, H13H-2433). Inhibitor HSA Binding [%] PSMA-1007 97.8DCFPyL 14.3 ⁶⁸Ga-^(nat)F-rhPSMA7-rac 96.7 ^(nat)Ga-¹⁸F-rhPSMA7.1 97.7^(nat)Ga-¹⁸F-rhPSMA7.2 97.8 ^(nat)Ga-¹⁸F-rhPSMA.3 96.9^(nat)Ga-¹⁸F-rhPSMA7.4 96.6

Biodistribution of [¹⁸F][^(nat)Ga]rhPSMA7.1-7.4 at 1 h pi

TABLE 10 Biodistribution (in % ID/g) of ¹⁸F-rhPSMAs at 1 h p.i in LNCaPtumor-bearing SCID mice. Data are expressed as mean ± SD (n = 4 forrhPSMA7.1, n = 5 for 7.2, n = 4 for 7.3, n = 5 for 7.4 and n = 3 for7-rac). [¹⁸F][^(nat)Ga]- [¹⁸F][^(nat)Ga]- [¹⁸F][^(nat)Ga]-[¹⁸F][^(nat)Ga]- [¹⁸F][^(nat)Ga]- rhPSMA-7-1 rhPSMA-7-2 rhPSMA-7-3rhPSMA-7-4 rhPSMA-rac blood 0.53 ± 0.13 0.56 ± 0.20 0.96 ± 0.24 1.15 ±0.30  1.1 ± 0.03 heart 0.53 ± 0.03 0.32 ± 0.13 0.87 ± 0.17 0.71 ± 0.260.69 ± 0.07 lung  1.1 ± 0.21 0.89 ± 0.38  2.2 ± 0.35 1.59 ± 0.61  1.4 ±0.17 liver 0.75 ± 0.62 0.35 ± 0.08 0.69 ± 0.13 0.69 ± 0.20 0.67 ± 0.07spleen 20.0 ± 4.2  10.1 ± 6.3  16.6 ± 2.6  18.4 ± 9.77 11.1 ± 2.3 pancreas 0.45 ± 0.12 0.21 ± 0.08 0.63 ± 0.44 0.50 ± 0.30 0.60 ± 0.10stomach 0.28 ± 0.17 0.19 ± 0.08 0.44 ± 0.23 0.25 ± 0.06 0.49 ± 0.07intestine 0.30 ± 0.16 0.18 ± 0.07 0.35 ± 0.07 0.37 ± 0.09 0.60 ± 0.27kidneys  220 ± 24.8 87.6 ± 28.8  292 ± 45.1  153 ± 80.3 71.3 ± 13.3adrenals  2.0 ± 0.25 1.3 ± 0.8  2.2 ± 0.83 3.57 ± 2.38  3.0 ± 0.45muscle 0.32 ± 0.30 0.13 ± 0.07 0.33 ± 0.15 0.31 ± 0.08 0.36 ± 0.06 bone0.50 ± 0.31 0.31 ± 0.24 0.38 ± 0.32 0.62 ± 0.30 0.91 ± 0.11 tumor 14.1 ±4.1  6.5 ± 2.3 18.3 ± 7.2  18.9 ± 3.27 10.4 ± 0.67

Biodistribution of [¹⁸F][^(nat)Ga]rhPSMA7.1-7.4 at 1 h pi withCompetition

TABLE 11 Biodistribution [% ID/g] of ¹⁸F-labeled rhPSMA tracersco-injected with PMPA (8 mg/kg) at 1 h p.i in LNCaP tumor-bearing SCIDmice. Data are expressed as mean ± SD (n = 3). [¹⁸F][^(nat)Ga]-[¹⁸F][^(nat)Ga]- [¹⁸F][^(nat)Ga]- [¹⁸F][^(nat)Ga]- rhPSMA-7-1 rhPSMA-7-2rhPSMA-7-3 rhPSMA-7-4 blood 0.86 ± 0.40  1.1 ± 0.31 0.55 ± 0.14 0.82 ±0.17 heart 0.37 ± 0.16 0.47 ± 0.09 0.26 ± 0.04 0.37 ± 0.05 lung 0.85 ±0.29  1.1 ± 0.32 0.69 ± 0.10 0.74 ± 0.14 liver 0.43 ± 0.07 0.46 ± 0.070.46 ± 0.14 0.48 ± 0.14 spleen 0.21 ± 0.08 0.26 ± 0.07 0.35 ± 0.02 0.28± 0.15 pancreas 0.16 ± 0.10 0.12 ± 0.05 0.11 ± 0.02 0.18 ± 0.09 stomach0.97 ± 0.81 0.21 ± 0.06 0.76 ± 0.74 0.20 ± 0.07 intestine 0.66 ± 0.320.33 ± 0.10 0.94 ± 0.97 0.36 ± 0.08 kidneys 10.9 ± 2.5  10.9 ± 1.0  15.5± 2.2  7.2 ± 2.4 adrenals 0.003 ± 0.004 0.07 ± 0.10 0.07 ± 0.09 0.03 ±0.04 muscle 0.17 ± 0.15 0.09 ± 0.03 0.09 ± 0.02 0.20 ± 0.05 bone 0.33 ±0.24 0.57 ± 0.39 0.34 ± 0.22 1.0 ± 0.8 tumor 0.94 ± 0.22  1.0 ± 0.13 1.5± 0.4 0.99 ± 0.19

Quantification of Relative Changes of the Amount of Each rhPSMA7.xIsomer in Blood, Kindey, Liver, Urine and Tumor After Application of[¹⁸F]rhPSMA7-rac

With the aim to quantify the relative changes of each rhPSMA7 isomer inblood, liver, kidney, urine and tumor 30 min after injection of[¹⁸F]rhPSMA7-rac into a LNCaP tumor bearing mouse, two differenthomogenization methods (a potter and a ball mill) were used to extractthe tracer from kidney, liver and tumor tissue (see Materials andMethods).

Table 12 summarizes the observed efficiencies for both homogenizationmethods and the efficancy of the subsequent solid phase extractionprocedure (to separate the tracer from the protein fraction).

TABLE 12 Determination of the decay corrected extracted activities fromthe examined tissue samples via the Potter-Elvehjem tissue grinder (n= 1) and the MM-400 ball mill (n = 3). Potter-Elvehjem tissue grinder (n= 1): EFFICIENCY [%] Sample SPE cartridge- extraction loading overallblood 93 93 86 kidney 91 66 60 tumor 90 59 53 MM-400 ball mill (n = 3):EFFICIENCY [%] blood 98 ± 2  94 ± 2 92 ± 3  liver 97 89 ± 2 86 ± 2 kidney 63 ± 5  68 ± 8 43 ± 8  tumor 64 ± 18 65 ± 3 42 ± 14

Whereas the extraction of activity from the samples using the potter wasquite efficient, the use of the ball mill was disappointing.Nevertheless, even with the ball mill >60% extraction efficiency wasreached.

Taking into account the possible species that could be formed bymetabolic cleavage of amide bonds of rhPSMA7, only a) species withsignificantly increase lipophilicity of b) F18-fluoride seem probable.Thus in principle it seem possible that “iL” species depicted in FIG. 12are not extracted from tissue sample (aqueous extraction) and thus donot appear in the final analysis. However, it should be noted that suchspecies would appear in vivo in the liver and intestine (hepatobiliaryexcretion of lipophilic compounds) or should be bound to plasma proteins(resulting in high activity levels for the blood, which on the otherhand showed excellent extraction efficiency).

For quantification of each isomer in the racemic mixture and especiallyfor the poorly separated first and second peak (rhPSMA 7.2 andrhPSMA7.3) a graphical approximation was initially used. This approachwas based on the assumption that a) each isomer is eluted from the HPLCcolumn with an identical peak shape and b) the different peak heightscan be used as first approximation to calculate by means of linearfactors less separated peaks (i.e. rhPSMA 7.2 and rhPSMA7.3).

Based on these assumptions, the first analysis was performed by usingone LNCaP tumor bearing mice coinfected with [¹⁸F][^(nat)Ga]rhPSMA7-rac.With the aim to validate these experiments by means of three additionalexperiments and to improve the graphical analyses by a more validprocedure, the Systat softare package ‘PeakFit’ was used. PeakFit allowsfor automated nonlinear separation, analysis and quantification of HPLCelution profiles by deconvolution procedures that uses a Gaussianresponse function with a Fourier deconvolution/filtering algorithm(https://systatsoftware.com/products/peakfit/).

A comparison of the graphical analysis of the first experiments revealedthat the graphical analysis overestimated the second peak (rhPSMA7.3),whereas the first peak was underestimated. Consequently, all data setswere reanalyzed and quantified by means of PeakFit.

HPLC-Analyses of 4 Independent Experiments in Tumor Bearing Mice 30 minp.i.

1. Evaluation of Peak 3 and 4 (rhPSMA7.4 and rhPSMA 7.1) by Radio-HPLC

It was first examined, whether the deconvolution technique shows similardata for the last two peaks (rhPSMA7.4 and 7.1) that have a goodseparation (although they are not baseline separated).

2. Evaluation of All Peaks (rhPSMA7.1, 7.2, 7.3 and 7.4) by Radio-HPLC

FIGS. 14a and 14b summarise the percentage change of each rhPSAM7.nisomer in a given sample with respect to its percentage in the injectedsolution ([¹⁸F][^(nat)Ga]rhPSMA7-rac); the results for the individualexperiment are shown in FIG. 14. The proportion of each isomer wasquantified by analysis of the HPLC elution profile by Systat ‘PeakFit’.Subsequently, the percentage change of each isomer in a given samplewith repect to its percentage in the injected solution was calculated.

3. Discussion of the HPLC Data

The radio-HPLC analyses of the radioactivity extracted from thehomogenized (kidney, liver, tumor) or diluted (blood) tissues andsubsequently immobilized on and eluted from the solid phase extractioncartridge did show no signs of metabolic instability. Thus, nolipophilic metabolic fragments were observed. It should be noted thatF-18-fluoride cannot be accurately detected by HPLC under the conditionsused for sample preparation (see TLC analysis).

Although there is a clear trend towards the D-Dap-derivative rhPSMA7.1and 7.3, the overall changes are low (max 15%). It is also important tostress in this context, that FIGS. 15 and 16 show “relative changes”without taking the absolute uptake values into account.

Although rhPSMA7.1 has the weakest affinity and internalization of allrhPSMA7 compounds, it shows the largest positive percentage change inblood liver, kidney and tumor.

Although the reason for this result is unclear, one can speculate thathomogenization of the tissue samples, even with the ball mill, did notresulted in a quantitative cell disruption. Thus, the rhPSMA7 tracerswith the highest internalization (rhPSMA7.2: 191.83%±15.54%, rhPSMA7.4:207.33±4.06% and rhPSMA7.3: 161.41%±8.88%) might have been extracted ina less efficient manner, whereas rhPSMA7.1 with its low internalizationof only 69.55%±5.29% was efficiently extracted and is consequentlyoverestimated in the HPLC analysis.

In addition, it seems that the rhPSMA compounds 7.2 and 7.4 are somewhatmore rapidly excreted (see values for urine). These compounds showgenerally negative changes in solid tissues and blood, although bothcompounds exhibit higher affinities and internalization rates whencompared with rhPSMA7.1. Whether this might be caused by metabolicdegradation of 7.2 and 7.4 (both are L-Dap derivative) is unclear, sinceno metabolites, i.e. lyophilic metabolites have been detected. It mighthowever be possible, that such metabolites (see FIG. 11), due to theirhigh logP value, are not extractable in aqueous buffer solutions. Inthis case, they should appear in the liver (see biodistribution) andperhaps in blood samples (high probability for high serum proteinbinding). Since no elevated activity accumulation has been observed forliver tissue in the course of the biodistribution studies and theactivity extraction from blood was highly efficient (see table 3), weassume that no significant degradation for rhPSMA7.2 and 7.4 occurred.This assumption is supported by unsuspicious SUV-values for liver tissue(gall bladder, intestine) in the context of the clinical use of[¹⁸F]rhpsma-rac in humans.

TLC-Analysis in Tumor Bearing Mice 30 min p.i.

Radio-TLC Analysis was carried out a) on urine samples by directlysubjecting a small volume onto a TLC strip, b) by analysis of a smallvolume of the non-immobilized activity during the SPE process (the‘breakthrough fraction’), and c) by analysis of a small volume of thecartridge eluates.

TABLE 13 TLC analysis of blood, organ and urine samples Comment * TLCScanner Phosphoimager TLC signal Intact Intact intensity [cts] tracer¹⁸F-Fluoride tracer ¹⁸F-Fluoride (overall very Date Sample [%] [%] [%][%] low-low) Jul. 30, QK 2018 Blood

Liver

Kidney 94.04 5.96 Methodological 369 problems Urine 82.51 17.49 726Tumor

Aug. 1, QK 94.27 5.73 96.02   3.98  384 2018 Blood

Liver

93.92^(#)   6.084^(#)

Kidney 94.58 5.42 572 Urine 96.2 3.80 98.55   1.55  395 Tumor

 

Aug. 2, QK Activity level 97.43   2.57  2016 too low for TLC   Blood96.80   3.20  Liver 74.15^(#) 25.85^(#) Kidney 96.48   3.52  Urine95.85   4.15  Tumor 96.47   4.15  (*) due to the low activity level, theTLC measurements with signal intensity < 200 cts have been deleted.

Discussion of the TLC Data

Since it is very difficult to detect n.c.a. ¹⁸F-fluoride by means ofRP-18 chromatography (due to free Si—OH groups of the matrix thatinteract with nca fluoride), thin layer chromatography was performed toinvestigate to quantify F-18-Fluoride in the extracted solutions.

Since none of the reagents and salts normally used for proteinprecipitation are tested for cold fluoride and to avoid possibleliberation of F-18-fluoride from the tracer by isotopic exchange,protein precipitation was not implemented in the sample preparationprocess—although such protein load often result in limited peakseparation, peak tailing and activity that sticks at the start line. Thesolutions obtained after tissue extraction (or blood centrifugation)were directly used for TLC analysis.

Although the activity available for analysis was quite low in allsamples, the TLC results reveal that the overall content of F-18-fluridewas below approx. 6% in the tissue investigated, except:

-   -   the urine sample obtained on Jul. 30, 2018 (17.49% free        fluoride),    -   the liver sample obtained on Aug. 2, 2018 (25.85% free        fluoride).

Whereas the analysis of the urine by TLC is regarded as valid result(see Profile in the FIG. 20), the result obtained with the liver sampleis caused by extensive tailing of the peak representing the intacttracer (see FIG. 18). In addition it can be concluded that the abovementioned max. 6% free fluoride represent an overestimation, since peaktailing, even obtained during the QK and release of [F-18]rhPSMA7-rac inPBS (FIG. 18) for clinical application show a tailing of the productpeak. As demonstrated by the phosphoimages, this tailing is observed inalmost every TLC analysis and contributes to the integrated area ofF-18-fluoride.

It need to be noted that neither the biodistribution studies, nor theclinical PET scans in humans (status July 2018: approx 1400 scans with[F-18]rhPSMA7-rac) resulted in any suspicious or identifiableF-18-acculuation in bone by liberated F-18-fluoride.

To further investigate the liberation of F-18 fluoride from[F-18]rhPSMA7-rac (as observed in one urine sample) we investigated theoccurrence of F-18-Fluoride in further urine samples (normal mice) bymeans of RP-18 HPLC (new RP-18 end-capped column) and TLC analyses.

Radio-TLC-Analysis of the Formation of F-18-Fluoride in Normal Mice 30min p.i.

For this purpose normal mice were used. Urine samples were collected bymeans of a catheter over a period of 30 min. The urine was centrifugedand directly subjected to HPLC and TLC.

As shown in FIG. 21, left column, free F-18-fluoride was found in urinesamples of all isomers and is also formed when fresh urine is “incubatedwith [F-18]rhPSMA7.4. (right column). Identification of F-18-fluoridewas performed by demonstrating that a) this species is retained on QMAcartridges (data not shown), b) is eluted in the dead volume of RP-18columns and c) can not be retained or mobilized on RP-18 columns orRP-18 TLC plates, respectively, irrespective of the mobile phases used.

Due to the fact that such high amounts of F-18 fluoride were notdetected in the HPLC analyses of blood or organs, such as kidneys,tumor, liver etc., that no elevated activity uptake in bone was observedin the biodistribution studies in mice and no elevated activity uptakein bone was observed during the clinical PET scans with the[F-18]rhPSMA7-rac compound since [F-18]rhPSMA7-rac has been establishedfor clinical scanning end of 2017 at the TUM (status end July, 2018:approx. 1400 PET scans in patients with prostate cancer) we concludedthat [F-18]fluoride might be formed downstream from glomerularfiltration of the tracer, resulting in the formation and subsequentexcretion of [F-18]fluoride WITHOUT detectable uptake of F-18-fluoridein blood, organs or bones.

This assumption is supported by the literature on the toxicology offluoride that describes relevant amounts of fluoride in KIDNEYS ANDURINE. Normal urinary fluoride levels of 0.3 ppm were observed in mice(Bouaziz H et al., Fluoride 2005; 38(1):23-31). In another publication,the average fluoride concentration in the urine of normal mice wasdetermined to be 0.13-0.14 μg/mL (Poesina N D et al. Rom J MorpholEmbryol 2014, 55(2):343-349), and Inkielewicz I. et al. found that thefluoride content in the serum of rats is about 5% of the concentrationof fluoride in the kidneys (serum: 0.051 μg/mL, kidneys: 0.942 μg/mL)(Fluoride; 36 (4); 263-266). Taken into account that most of the traceris specifically taken up into and also physiologically cleared by thekidneys, an elevated fluoride level in the kidney, combined with a bodytemperature of 36.6° C., might result in a continuous elimination ofF-18-fluoride from the rhPSMA-compounds in kidneys.

Consequently, fresh and nonradioactive urine samples collected fromnormal mice were incubated with [F-18]rhPSMA7.x for various time periods(see legend to FIG. 21). FIG. 22, right column, clearly demonstrate thatincubation of urine with [F-18]rhPSMA7.x EX VIVO result in the formationof free [F-18]fluoride to various degrees, promoted by the differentconcentrations of cold F-19-Fluoride in the urine samples and increasingover time.

To further support the hypothesis, 500 nmol cold F-19-fluoride was addedto fresh and nonradioactive urine of mice, followed by the addition of[F-18]rhPSMA7.3 and incubation for 2 h. According to the hypothesis, thehigh concentration of [F-19]fluoride should result in the formation of asignificant amount of [F-18]fluoride. FIG. 23 shows that under theseconditions 98.5% of the radioactivity is exchanged and forms[F-18]fluoride within 2 h (FIG. 23).

Since isotopic exchange rates are depending on the concentration of thefour relevant species in the equilibrium ([F-18]Fluoride,[F-19]fluoride, [F-18]rhPSMA7.3 and [F-19]rhPSMA7.3), it was alsoinvestigated, whether the addition of [F-18]fluoride to fresh andradioactive urine (20.6% [F-18]Fluoride, 79.4% [F-18]rhpsma7.3) followedby the addition of cold [F-19]rhPSMA7.3 tracer also result in thelabeling of the radiopharmaceutical [F-18]rhPSMA7-3. Unexpectedly, evena small amount of 5 nmol [F-19]rhPSMA7-3 to the urine above resulted inan increase of [F-18]rhPSMA7.3 from 79.4% to 85.8% (F-18]Fluoridedecreased from 20.6% to 14.2%) at room temperature.

The results obtained by isotopic exchange in urine are consideredrepresentative for all tracers conjugated with the4-(di-tert-butyl[(18)F]fluorosilyl)-benzyl)oxy moiety and thus for allrhPSAM7 isomers.

Preclinical Dosimetry, Human Biodistribution and Uptake in Tumor Lesions

Please note that in the following 18F-rhPSMA-7 refers to^(nat)Ga-¹⁸F-rhPSMA7-rac and 18F-rhPSMA-7.3 to ^(nat)Ga-¹⁸F-rhPSMA7.3

A) Preclinical Dosimetry of 18F-rhPSMA-7 and 18F-rhPSMA-7.3 in Mice

Aim was to assess the distribution and excretion of ¹⁸F-rhPSMA-7 and¹⁸F-rhPSMA-7.3 at different time-points up to 300 minutes following asingle intravenous administration in mice and to perform calculationsfor internal dosimetry.

Methods

3-5 mice were injected per timepoint with a mean 25.6±3.6 MBq of18F-rhPSMA-7 and 28.5±4.8 MBq of 18F-rhPSMA-7.3, respectively. Mice,severe combined immunodeficiency (SCID) were used for the experiments.All animal experiments were conducted in accordance with general animalwelfare regulations in Germany and the institutional guidelines for thecare and use of animals.

Mice were sacrificed at the following timepoints:

¹⁸F-rhPSMA-7: 10, 20, 40, 60, 120 and 180 minutes after administration.

¹⁸F-rhPSMA-7.3: 10, 60, 120, 180 and 300 minutes after administration.

Please note that based on initial experiments exhibiting prolonged renalkidney uptake for ¹⁸F-rhPSMA-7.3 a late timepoint (300 min) was used forthe final experiments.

The following tissues/fluids were harvested:

Urine, blood, heart, lung, spleen, pancreas, liver, stomach (emptied),small intestine (emptied), large intestine (emptied), kidneys, bladder,testis, fat, muscle (partial, femoral), femur, tail and brain. Urine wascollected with a pipette in the CO₂ gas chamber. In case of missingurination in the chamber the bladder was aspirated with an insulinsyringe. Blood was withdrawn instantly after sacrifice with an insulinsyringe from the heart. All other tissues and organs were dissected andtransferred directly in plastic containers.

The weights of the samples in the plastic containers were measured usingan electronic balance. The weights of the empty and pre-labeled plasticcontainers for the dedicated samples were measured beforehand. The tareweight of the plastic containers was subtracted from the weight of themeasurement sample with the plastic container. The thus-calculatedweight was designated as the weight of the measurement sample.

The plastic containers containing the measurement samples were placed inspecific racks of an automatic gamma counter (PerkinElmer-Wallac,Waltham, USA) for measuring the counting rate over 60 seconds (countsper minute=cpm). In addition, a 1% (v/v) standard (n=5) with a knownamount of radioactivity was measured together with the samples toconvert the counting rate of the organ samples into activity.

Data Analysis

The counting rates of measurement samples were automatically correctedfor decay. The radioactivity distribution ratios (unit: percentage ofthe injected dose (% ID) in the measurement samples were determinedusing the equation below. The sum of the counting rates from allmeasurement samples obtained from one mouse was designated as thecounting rate for administrated radioactivity.

${{Percentage}\mspace{14mu}{of}\mspace{14mu}{injected}\mspace{14mu}{{dose}\left( {\%\mspace{14mu} I\; D} \right)}} = {\frac{\begin{matrix}{{{counting}\mspace{14mu}{rate}\mspace{14mu}{for}}\mspace{14mu}} \\{{measurement}\mspace{14mu}{sample}}\end{matrix}}{\begin{matrix}{{{sum}\mspace{14mu}{of}\mspace{14mu}{counting}\mspace{14mu}{rates}}\mspace{14mu}} \\{{{for}\mspace{14mu}{all}\mspace{14mu}{measurement}}\mspace{14mu}} \\{{samples}\mspace{14mu}{from}\mspace{14mu}{one}\mspace{14mu}{mouse}}\end{matrix}}*100}$

The radioactivity distribution ratio per unit weight of the measurementsample (unit: % ID/g) excluding urine and feces samples was determinedby using the equation below. The weight of the measurement sample wasdetermined by subtraction of the empty measurement container from thecontainer including the sample.

${{Percentage}\mspace{14mu}{of}\mspace{14mu}{injected}\mspace{14mu}{{dose}\left( {\%\mspace{14mu} I\;{D/g}} \right)}} = \frac{{Percentage}\mspace{14mu}{of}\mspace{14mu}{injected}\mspace{14mu}{{dose}\left( {\%\mspace{14mu} I\; D} \right)}}{{weight}\mspace{14mu}{of}\mspace{14mu}{measurement}\mspace{14mu}{{sample}(g)}}$

Dosimetry Analysis

For consistency of statistical calculations for each radiotracer thesame number of time-points for ¹⁸F-rhPSMA-7 and ¹⁸F-rhPSMA-7.3 was used.Therefore, for ¹⁸F-rhPSMA-7 the 10 min and 20 min time points werecombined creating a 15 min endpoint.

The time-integral of activity for the accumulation in the significantsource organs (AUCs) were generated both with numerical integration andphysical decay according to J Juan et al., Journal of PharmaceuticalSciences, 1993, 82:762-763.

Kirshner et al. established a method that uses linear scaling of thepercent injected dose in the animal by the ratio of the organ weightsand total body weights of phantoms in both species.

-   -   Kirschner A S, Ice R D, Beierwaltes W H. Radiation Dosimetry of        131I-19-Iodocholesterol. J Nucl Med. 1973 Sep. 1; 14(9):713-7.    -   Kirschner A, Ice R, Beierwaltes W. Letters to the editor. J Nucl        Med. (1975):248-9.

In brief, to calculate a human dosimetry from the biodistribution in themice, an extrapolation was necessary to account for the differencesbetween the animals and humans. Normal-organ radiation doses wereestimated for the 70-kg Standard Adult anatomic model usingtime-depending organ activity concentrations (in percent of the injecteddose per gram, % ID/g) and total-body activities measured in thebiodistribution studies in mice.

Tissue activity concentrations in mice were converted to tissuefractional activities in the 70-kg Standard Adult using the relativefractional organ masses in the Standard Adult and the “standard”25-gramm mouse. Time-dependent total-body activity was fit to anexponential function and the difference between the injected activityand the total-body activity was assumed to be excreted to the urinebecause activity concentrations in the liver and GI tracer were low atall time points studied.

Organ residence time was calculated by numerical integration using thetrapezoidal rule and the rest-of-body ¹⁸F residence times was calculatedas the difference between the total-body residence time and the sum ofthe organ and urine residence times. The bladder contents residence timewas estimated using the dynamic voiding model in the OLINDA/EXM 1.0dosimetry software. Finally, the Standard Adult mean organ doseequivalents (in mSv/MBq) and effective dose (also in mSv/MBq) were thencalculated using OLINDA/EXM 1.0.

Final calculation of radiation absorbed dose and dosimetry frombiodistribution in mice: The tissues or organs in which a significantaccumulation of radioactivity occurs (i.e., source organ) were kidney,spleen, lung, liver and heart. With respect to activity accumulation andclearance, a rapid clearance from blood and clearance to urine butrelatively slow build-up in kidney was found.

Results

TABLE 14 Dosimetry results for ¹⁸F-rhPSMA-7 using a 3.5 h bladdervoiding interval. Target Organ Alpha Beta Photon Total EDE Cont. EDCont. Adrenals 0.00E000 1.95E−03 5.85E−03 7.80E−03 0.00E000 3.90E−05Brain 0.00E000 1.95E−03 2.54E−03 4.49E−03 0.00E000 2.24E−05 Breasts0.00E000 1.95E−03 2.29E−03 4.24E−03 6.36E−04 2.12E−04 Gallbladder Wall0.00E000 1.95E−03 5.54E−03 7.49E−03 0.00E000 0.00E000 LLI Wall 0.00E0001.95E−03 1.41E−02 1.61E−02 9.66E−04 1.93E−03 Small Intestine 0.00E0001.95E−03 8.40E−03 1.04E−02 0.00E000 5.18E−05 Stomach Wall 0.00E0001.95E−03 5.05E−03 7.00E−03 0.00E000 8.40E−04 ULI Wall 0.00E000 1.95E−037.31E−03 9.26E−03 0.00E000 4.63E−05 Heart Wall 0.00E000 8.82E−043.54E−03 4.42E−03 0.00E000 0.00E000 Kidneys 0.00E000 4.70E−02 1.80E−026.51E−02 3.91E−03 3.25E−04 Liver 0.00E000 6.35E−04 3.63E−03 4.27E−030.00E000 2.13E−04 Lungs 0.00E000 1.25E−03 3.04E−03 4.30E−03 5.16E−045.16E−04 Muscle 0.00E000 1.95E−03 5.73E−03 7.68E−03 0.00E000 3.84E−05Ovaries 0.00E000 1.95E−03 1.35E−02 1.55E−02 3.87E−03 3.09E−03 Pancreas0.00E000 1.95E−03 6.13E−03 8.08E−03 0.00E000 4.04E−05 Red Marrow0.00E000 1.39E−03 5.41E−03 6.80E−03 8.16E−04 8.16E−04 Osteogenic Cells0.00E000 4.18E−03 4.75E−03 8.93E−03 2.68E−04 8.93E−05 Skin 0.00E0001.95E−03 2.98E−03 4.93E−03 0.00E000 4.93E−05 Spleen 0.00E000 1.59E−021.01E−02 2.61E−02 1.56E−03 1.30E−04 Testes 0.00E000 1.95E−03 9.69E−031.16E−02 0.00E000 0.00E000 Thymus 0.00E000 1.95E−03 3.24E−03 5.18E−030.00E000 2.59E−05 Thyroid 0.00E000 1.95E−03 3.23E−03 5.18E−03 1.55E−042.59E−04 Urinary Bladder Wall 0.00E000 2.45E−01 1.08E−01 3.54E−012.12E−02 1.77E−02 Uterus 0.00E000 1.95E−03 2.61E−02 2.80E−02 1.68E−031.40E−04 Total Body 0.00E000 2.37E−03 5.39E−03 7.77E−03 0.00E0000.00E000 Effective Dose Equivalent (mSv/MBq) 3.56E−02 Effective Dose(mSv/MBq) 2.66E−02

TABLE 15 Dosimetry results for ¹⁸F-rhPSMA-7 using a 1.0 h bladdervoiding interval. Target Organ Alpha Beta Photon Total EDE Cont. EDCont. Adrenals 0.00E000 1.95E−03 5.64E−03 7.59E−03 0.00E000 3.79E−05Brain 0.00E000 1.95E−03 2.54E−03 4.49E−03 0.00E000 2.24E−05 Breasts0.00E000 1.95E−03 2.25E−03 4.20E−03 6.30E−04 2.10E−04 Gallbladder Wall0.00E000 1.95E−03 4.96E−03 6.91E−03 0.00E000 0.00E000 LLI Wall 0.00E0001.95E−03 7.25E−03 9.20E−03 5.52E−04 1.10E−03 Small Intestine 0.00E0001.95E−03 5.79E−03 7.73E−03 0.00E000 3.87E−05 Stomach Wall 0.00E0001.95E−03 4.70E−03 6.65E−03 0.00E000 7.98E−04 ULI Wall 0.00E000 1.95E−035.33E−03 7.27E−03 0.00E000 3.64E−05 Heart Wall 0.00E000 8.82E−043.48E−03 4.36E−03 0.00E000 0.00E000 Kidneys 0.00E000 4.70E−02 1.76E−026.47E−02 3.88E−03 3.23E−04 Liver 0.00E000 6.35E−04 3.39E−03 4.02E−030.00E000 2.01E−04 Lungs 0.00E000 1.25E−03 3.01E−03 4.26E−03 5.11E−045.11E−04 Muscle 0.00E000 1.95E−03 4.01E−03 5.96E−03 0.00E000 2.98E−05Ovaries 0.00E000 1.95E−03 7.21E−03 9.16E−03 2.29E−03 1.83E−03 Pancreas0.00E000 1.95E−03 5.86E−03 7.80E−03 0.00E000 3.90E−05 Red Marrow0.00E000 1.39E−03 4.29E−03 5.68E−03 6.82E−04 6.82E−04 Osteogenic Cells0.00E000 4.18E−03 4.09E−03 8.27E−03 2.48E−04 8.27E−05 Skin 0.00E0001.95E−03 2.38E−03 4.32E−03 0.00E000 4.32E−05 Spleen 0.00E000 1.59E−029.90E−03 2.58E−02 1.55E−03 1.29E−04 Testes 0.00E000 1.95E−03 5.09E−037.03E−03 0.00E000 0.00E000 Thymus 0.00E000 1.95E−03 3.20E−03 5.15E−030.00E000 2.57E−05 Thyroid 0.00E000 1.95E−03 3.23E−03 5.17E−03 1.55E−042.59E−04 Urinary Bladder Wall 0.00E000 7.87E−02 3.66E−02 1.15 E−016.92E−03 5.76E−03 Uterus 0.00E000 1.95E−03 1.12E−02 1.31E−02 7.87E−046.56E−05 Total Body 0.00E000 2.27E−03 3.93E−03 6.19E−03 0.00E0000.00E000 Effective Dose Equivalent (mSv/MBq) 1.82E−02 Effective Dose(mSv/MBq) 1.22E−02

TABLE 16 Dosimetry results for ¹⁸F-rhPSMA-7.3 using a 3.5 h bladdervoiding interval. Target Organ Alpha Beta Photon Total EDE Cont. EDCont. Adrenals 0.00E000 3.12E−03 7.93E−03 1.10E−02 0.00E000 5.52E−05Brain 0.00E000 3.12E−03 4.07E−03 7.19E−03 0.00E000 3.59E−05 Breasts0.00E000 3.12E−03 3.55E−03 6.67E−03 1.00E−03 3.34E−04 Gallbladder Wall0.00E000 3.12E−03 7.46E−03 1.06E−02 0.00E000 0.00E000 LLI Wall 0.00E0003.12E−03 1.28E−02 1.59E−02 9.53E−04 1.91E−03 Small Intestine 0.00E0003.12E−03 9.42E−03 1.25E−02 0.00E000 6.27E−05 Stomach Wall 0.00E0003.12E−03 7.02E−03 1.01E−02 0.00E000 1.22E−03 ULI Wall 0.00E000 3.12E−038.57E−03 1.17E−02 0.00E000 5.85E−05 Heart Wall 0.00E000 1.32E−035.39E−03 6.71E−03 0.00E000 0.00E000 Kidneys 0.00E000 5.11E−02 2.07E−027.18E−02 4.31E−03 3.59E−04 Liver 0.00E000 9.70E−04 5.02E−03 5.99E−030.00E000 3.00E−04 Lungs 0.00E000 1.95E−03 4.66E−03 6.61E−03 7.93E−047.93E−04 Muscle 0.00E000 3.12E−03 6.55E−03 9.67E−03 0.00E000 4.83E−05Ovaries 0.00E000 3.12E−03 1.26E−02 1.57E−02 3.92E−03 3.14E−03 Pancreas0.00E000 3.12E−03 8.29E−03 1.14E−02 0.00E000 5.70E−05 Red Marrow0.00E000 2.22E−03 6.79E−03 9.01E−03 1.08E−03 1.08E−03 Osteogenic Cells0.00E000 6.70E−03 6.52E−03 1.32E−02 3.97E−04 1.32E−04 Skin 0.00E0003.12E−03 3.83E−03 6.95E−03 0.00E000 6.95E−05 Spleen 0.00E000 1.52E−021.15E−02 2.67E−02 1.60E−03 1.34E−04 Testes 0.00E000 3.12E−03 8.96E−031.21E−02 0.00E000 0.00E000 Thymus 0.00E000 3.12E−03 5.08E−03 8.20E−030.00E000 4.10E−05 Thyroid 0.00E000 3.12E−03 5.15E−03 8.27E−03 2.48E−044.14E−04 Urinary Bladder Wall 0.00E000 1.56E−01 7.14E−02 2.27E−011.36E−02 1.14E−02 Uterus 0.00E000 3.12E−03 2.04E−02 2.36E−02 1.41E−031.18E−04 Total Body 0.00E000 3.52E−03 6.33E−03 9.86E−03 0.00E0000.00E000 Effective Dose Equivalent (mSv/MBq) 2.94E−02 Effective Dose(mSv/MBq) 2.17E−02

TABLE 17 Dosimetry results for ¹⁸F-rhPSMA-7.3 using a 1.0 h bladdervoiding interval. Target Organ Alpha Beta Photon Total EDE Cont. EDCont. Adrenals 0.00E000 3.12E−03 7.79E−03 1.09E−02 0.00E000 5.46E−05Brain 0.00E000 3.12E−03 4.06E−03 7.18E−03 0.00E000 3.59E−05 Breasts0.00E000 3.12E−03 3.53E−03 6.65E−03 9.97E−04 3.32E−04 Gallbladder Wall0.00E000 3.12E−03 7.11E−03 1.02E−02 0.00E000 0.00E000 LLI Wall 0.00E0003.12E−03 8.45E−03 1.16E−02 6.94E−04 1.39E−03 Small Intestine 0.00E0003.12E−03 7.78E−03 1.09E−02 0.00E000 5.45E−05 Stomach Wall 0.00E0003.12E−03 6.80E−03 9.92E−03 0.00E000 1.19E−03 ULI Wall 0.00E000 3.12E−037.33E−03 1.05E−02 0.00E000 5.23E−05 Heart Wall 0.00E000 1.32E−035.36E−03 6.68E−03 0.00E000 0.00E000 Kidneys 0.00E000 5.11E−02 2.04E−027.16E−02 4.29E−03 3.58E−04 Liver 0.00E000 9.70E−04 4.87E−03 5.84E−030.00E000 2.92E−04 Lungs 0.00E000 1.95E−03 4.63E−03 6.58E−03 7.90E−047.90E−04 Muscle 0.00E000 3.12E−03 5.47E−03 8.59E−03 0.00E000 4.30E−05Ovaries 0.00E000 3.12E−03 8.61E−03 1.17E−02 2.93E−03 2.35E−03 Pancreas0.00E000 3.12E−03 8.12E−03 1.12E−02 0.00E000 5.62E−05 Red Marrow0.00E000 2.22E−03 6.09E−03 8.31E−03 9.97E−04 9.97E−04 Osteogenic Cells0.00E000 6.70E−03 6.11E−03 1.28E−02 3.84E−04 1.28E−04 Skin 0.00E0003.12E−03 3.45E−03 6.57E−03 0.00E000 6.57E−05 Spleen 0.00E000 1.52E−021.14E−02 2.66E−02 1.59E−03 1.33E−04 Testes 0.00E000 3.12E−03 6.08E−039.20E−03 0.00E000 0.00E000 Thymus 0.00E000 3.12E−03 5.06E−03 8.18E−030.00E000 4.09E−05 Thyroid 0.00E000 3.12E−03 5.15E−03 8.27E−03 2.48E−044.13E−04 Urinary Bladder Wall 0.00E000 5.18E−02 2.66E−02 7.84E−024.70E−03 3.92E−03 Uterus 0.00E000 3.12E−03 1.11E−02 1.43E−02 8.55E−047.13E−05 Total Body 0.00E000 3.45E−03 5.42E−03 8.87E−03 0.00E0000.00E000 Effective Dose Equivalent (mSv/MBq) 1.85E−02 Effective Dose(mSv/MBq) 1.28E−02

CONCLUSION

The radioactivity distribution ratios were highest in kidneys afteradministration of both ¹⁸F-rhPSMA-7 and ¹⁸F-rhPSMA-7.3 at all examinedtime points in mice. Moreover, it was high in the spleen and in thebladder for both radiotracers compared to all other assessed tissues,where activity ratios were lower than 8% ID/g.

Since the majority of ¹⁸F-rhPSMA-7/¹⁸F-rhPSMA-7.3 activity augment inthe kidneys and the excretion via bladder reveal high activities, themain excretion route is defined via kidneys and the urinary system.

Using a 3.5 h and 1.0 h bladder voiding interval the extrapolated totaleffective doses were 2.66E−02 and 1.22E−02 mSv/MBq for ¹⁸F-rhPSMA-7 and2.17E−02 and 1.28E−02 mSv/MBq for ¹⁸F-rhPSMA-7.3, respectively. Aninjection of up to 370 MBq (10 mCi) for a clinical scan would result ina favorable radiation exposure of less than 5 mSv for both agentsassuming a 1h voiding interval.

Differences worth to mention between both radiotracers are only evidentregarding kidney uptake as ¹⁸F-rhPSMA-7.3 tends to accumulate moregradual with longer retention. Yet radiation exposure is comparablebetween both agents.

B) Human Biodistribution and Uptake in Tumor Lesions of 18F-rhPSMA-7 and18F-rhPSMA-7.3

The following sections describe biodistribution of 18F-rhPSMA-7 and18F-rhPSMA-7.3. Proof-of-concept evaluation was conducted undercompassionate use. The agent was applied in compliance with The GermanMedicinal Products Act, AMG § 13 2b, and in accordance with theresponsible regulatory body (Government of Oberbayern).

All subjects were examined on a Biograph mCT scanner (Siemens MedicalSolutions, Erlangen, Germany). All PET scans were acquired in 3D-modewith an acquisition time of 2-4 min per bed position. Emission data werecorrected for randoms, dead time, scatter, and attenuation and werereconstructed iteratively by an ordered-subsets expectation maximizationalgorithm (four iterations, eight subsets) followed by apostreconstruction smoothing Gaussian filter (5-mm full width atone-half maximum).

Methods

Human biodistribution was estimated by analysing clinical ¹⁸F-rhPSMA-7-and ¹⁸F-rhPSMA-7.3-PET/CT exams in 47 and 32 patients, respectively.Mean injected activities were 324 (range 236-424) MBq vs. 345 (range235-420) MBq and uptake times were 84 (range 42-166) min and vs. 76(range 59-122) min for ¹⁸F-rhPSMA-7 vs. ¹⁸F-rhPSMA-7.3, respectively.

The mean and maximum standardized uptake values (SUVmean/SUVmax) weredetermined for background (gluteal muscle), normal organs (salivaryglands, blood pool, lung, liver, spleen, pancreas, duodenum, kidney,bladder, bone) and three representative tumor lesions. Tumor uptake wasanalyzed in 89 lesions (26 primary tumors/local recurrences, 23 bone, 38lymph node and 2 visceral metastases) and 63 lesions (14 primarytumors/local recurrences, 30 bone, 18 lymph node and 1 visceralmetastases) for ¹⁸F-rhPSMA-7 and ¹⁸F-rhPSMA-7.3, respectively.

For calculation of the SUV, circular regions of interest were drawnaround areas with focally increased uptake in transaxial slices andautomatically adapted to a three-dimensional volume of interest (VOI) ata 50% isocontour. Organ-background and Tumor-background ratios werecalculated.

Results

Human biodistribution of ¹⁸F-rhPSMA-7 and ¹⁸F-rhPSMA-7.3 showed thetypical pattern known from other PSMA-ligands. Uptake parameters for¹⁸F-rhPSMA-7 and ¹⁸F-rhPSMA-7.3 were very similar with a lower activityretention in the bladder and higher uptake in tumor lesions for¹⁸F-rhPSMA-7.3: SUVmean for ¹⁸F-rhPSMA-7 vs. ¹⁸F-rhPSMA-7.3 were 16.9vs. 16.0 (parotid gland), 19.6 vs. 19.6 (submandibular gland), 2.0 vs.1.9 (blood pool), 0.7 vs. 0.7 (lungs), 7.0 vs. 7.3 (liver), 9.1 vs. 8.5(spleen), 32.4 vs. 35.5 (kidney), 2.5 vs. 2.8 (pancreas), 10.9 vs. 11.0(duodenum), 1.1 vs. 1.3 (non-diseased bone) and 10.2 vs. 2.0 (bladder)for ¹⁸F-rhPSMA-7 vs. ¹⁸F-rhPSMA-7.3, respectively. In particular, uptakevalues of ¹⁸F-rhPSMA-7.3 vs. ¹⁸F-rhPSMA-7 were significantly lower forretention in the bladder (2.0±0.8 vs. 6.3±21.2, p<0.05) andsignificantly higher for tumor lesions (32.5±42.7 vs. 20.0±20.2,p<0.05).

TABLE 18 SUVmax and SUVmean of normal organs and tumor lesions using¹⁸F-rhPSMA-7. Data are shown as mean, minimum and maximum. SUVmaxSUVmean mean min max mean min max background 1.0 0.6 1.8 0.6 0.4 1.2parotic gland 23.8 8.2 42.3 16.9 5.5 32.7 submandibular 27.0 10.1 43.819.6 7.0 29.7 gland bloodpool 2.4 1.6 3.9 2.0 1.1 17.0 lungs 1.1 0.5 3.10.7 0.3 2.0 liver 9.5 4.5 25.2 7.0 3.2 17.7 spleen 11.8 4.7 21.0 9.1 3.417.1 kidney 44.8 19.1 75.2 32.4 13.2 54.7 pancreas 3.7 1.8 7.9 2.5 1.35.5 duodenum 14.8 2.8 32.7 10.9 1.9 23.9 bone 1.7 0.8 3.1 1.1 0.6 2.1bladder 8.5 0.5 112.0 6.3 0.3 85.7 tumor 27.6 3.1 167.2 20.0 2.1 115.7

TABLE 19 SUVmax and SUVmean of normal organs and tumor lesions using¹⁸F-rhPSMA-7.3. Data are shown as mean, minimum and maximum. SUVmaxSUVmean mean min max mean min max background 1.0 0.6 1.7 0.7 0.4 1.1parotic gland 24.6 11.2 38.3 16.0 8.2 25.0 submandibular gland 28.4 14.647.4 19.6 10.4 33.4 bloodpool 2.8 1.9 3.9 1.8 1.3 2.5 lungs 1.1 0.7 1.90.7 0.4 1.1 liver 9.7 4.6 15.4 7.3 3.2 12.3 spleen 11.4 5.0 22.5 8.5 3.717.9 kidney 51.9 30.9 99.9 35.5 20.7 70.6 pancreas 4.2 2.4 7.8 2.8 1.65.2 duodenum 16.4 6.1 32.2 11.0 3.0 23.0 bone 2.1 1.1 3.4 1.3 0.7 2.2bladder 3.1 1.1 6.0 2.0 0.7 4.1 tumor 44.0 2.4 316.0 32.5 1.6 224.1

TABLE 20 Ratio SUVmax and SUVmean to background of normal organs andtumor lesions using ¹⁸F-rhPSMA-7. Data are shown as mean, minimum andmaximum. ratio SUVmax ratio SUVmean mean min max mean min max parotidgland 25.2 8.2 45.3 28.3 9.2 54.5 submandibular gland 28.7 10.1 54.733.3 11.7 61.8 bloodpool 2.5 1.3 4.8 3.2 1.6 21.3 lungs 1.1 0.4 3.3 1.10.4 4.0 liver 10.4 4.7 42.0 11.9 4.6 44.3 spleen 12.5 4.7 35.0 15.1 5.739.5 kidney 48.1 18.2 98.7 55.2 19.8 109.3 pancreas 3.9 1.5 11.3 4.3 1.910.8 duodenum 15.7 2.8 31.3 18.4 3.2 35.3 bone 1.7 0.9 2.9 1.8 1.0 3.2bladder 8.7 0.6 112.0 10.2 0.5 142.8 tumor 32.0 3.1 278.6 36.0 3.5 289.3

TABLE 21 Ratio SUVmax and SUVmean to background of normal organs andtumor lesions using ¹⁸F-rhPSMA-7.3. Data are shown as mean, minimum andmaximum. ratio SUVmax ratio SUVmean mean min max mean min max parotidgland 24.7 11.9 46.2 25.2 12.4 44.6 submandibular gland 28.2 14.0 62.130.6 15.7 62.3 bloodpool 2.8 1.5 5.2 2.9 1.7 4.9 lungs 1.0 0.6 1.8 1.00.6 1.8 liver 9.7 4.0 19.0 11.4 4.1 20.7 spleen 11.4 3.6 22.4 13.3 3.928.6 kidney 51.8 25.7 93.0 55.6 27.6 95.5 pancreas 4.1 2.2 6.9 4.4 2.37.8 duodenum 16.2 6.9 34.3 17.1 4.7 39.4 bone 2.0 1.1 3.2 2.1 1.0 3.6bladder 3.1 0.9 5.5 3.1 0.9 6.8 tumor 43.6 1.7 321.2 50.8 1.8 356.4

CONCLUSION

Human biodistribution is similar between ¹⁸F-rhPSMA-7 and ¹⁸F-rhPSMA-7.3for most normal organs. However, tracer retention in the bladder issignificantly lower and uptake in tumor lesions significantly higher for¹⁸F-rhPSMA-7.3 posing a clear advantage for clinical imaging. Imagingexamples with favorable human biodistribution and high uptake of tumorlesions of ¹⁸F-rhPSMA-7.3 are shown in FIG. 28.

1. A ligand-SIFA-chelator conjugate, comprising, within a singlemolecule three separate moieties: (a) one or more ligands which arecapable of binding to PSMA, (b) a silicon-fluoride acceptor (SIFA)moiety which comprises a covalent bond between a silicon and a ¹⁸Ffluorine atom, and (c) one or more chelating groups, containing achelated nonradioactive metal cation.
 2. The conjugate in accordancewith claim 1, wherein the silicon-fluoride acceptor (SIFA) moiety hasthe structure represented by formula (I):

wherein R^(1S) and R^(2S) are independently a linear or branched C3 toC10 alkyl group; R^(3S) is a C1 to C20 hydrocarbon group comprising oneor more aromatic and/or aliphatic units and/or up to 3 heteroatomsselected from O and S; and wherein the SIFA moiety is attached to theremainder of the conjugate via the bond marked by

.
 3. The conjugate in accordance with claim 2, wherein thesilicon-fluoride acceptor (SIFA) moiety has the structure represented byformula (Ia):

wherein t-Bu indicates a tert-butyl group.
 4. The conjugate inaccordance with any one of claims 1 to 3, wherein the chelating groupcomprises at least one of (i) a macrocyclic ring structure with 8 to 20ring atoms of which 2 or more are heteroatoms selected from oxygen atomsand nitrogen atoms; (ii) an acyclic, open chain chelating structure with8 to 20 main chain atoms of which 2 or more are heteroatoms selectedfrom oxygen atoms and nitrogen atoms; or (iii) a branched chelatingstructure containing a quaternary carbon atom.
 5. The conjugate inaccordance with claim 4, wherein the chelating group is selected frombis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a),cyclohexyl-1,2-diaminetetraacetic acid (CDTA),4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA),N′-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide(DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan(DO2A) 1,4,7,10-tetracyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA),α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTAGA), 1,4,7,10 tetraazacyclododecane N, N′, N″, N′″1,4,7,10-tetra(methylene) phosphonic acid (DOTMP),N,N′-dipyridoxylethylendiamine-N,N′-diacetate-5,5′-bis(phosphat) (DPDP),diethylene triamine N,N′,N″ penta(methylene) phosphonic acid (DTMP),diethylenetriaminepentaacetic acid (DTPA),ethylenediamine-N,N′-tetraacetic acid (EDTA),ethyleneglykol-O,O-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA),N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED),hydroxyethyldiaminetriacetic acid (H EDTA),1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate(HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), tetra3-hydroxy-N-methyl-2-pyridinone chelators(4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoicacid), abbreviated as Me-3,2-HOPO, 1,4,7-triazacyclononan-1-succinicacid-4,7-diacetic acid (NODASA),1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7-triazacyclononane(NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA),4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane(TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA),tris(hydroxypyridinone) (THP), terpyridin-bis(methyleneamintetraaceticacid (TMT),1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinicacid] (TRAP), 1,4,7,10-tetraazacyclotridecan-N,N′,N″,N′″-tetraaceticacid (TRITA),3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazonan-1-yl]methyl-hydroxy-phosphoryl]propanoicacid, and triethylenetetraaminehexaacetic acid (TTHA).
 6. The conjugatein accordance with any one of claims 1 to 5, wherein the chelating groupis 1,4,7,10-tetracyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA),α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTAGA) or1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinicacid] (TRAP).
 7. The conjugate in accordance with any one of claims 1 to6, wherein the chelator contains a chelated non-radioactive cationselected from the cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi,Ac, Th or Er.
 8. The conjugate in accordance with claim 7, wherein thechelator contains a chelated non-radioactive cation selected from thecations of Ga, or Lu.
 9. The conjugate in accordance with claim 8,wherein the chelator contains a chelated non-radioactive cation selectedfrom the cations of Ga³⁺.
 10. The conjugate in accordance with any oneof claims 1 to 9, which is a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein: SIFA is asilicon-fluoride acceptor (SIFA) moiety which comprises a covalent bondbetween a silicon and an ¹⁸F fluorine atom; m is an integer of 2 to 6; nis an integer of 2 to 6; R^(1L) is CH₂, NH or O R^(3L) is CH₂, NH or O;R^(2L) is C or P(OH); X¹ is selected from an amide bond, an ether bond,a thioether bond, an ester bond, a thioester bond, an urea bridge, andan amine bond; X² is selected from an amide bond, an ether bond, athioether bond, an ester bond, a thioester bond, an urea bridge, and anamine bond; L¹ is a divalent linking group with a structure selectedfrom an oligoamide, an oligoether, an oligothioether, an oligoester, anoligothioester, an oligourea, an oligo(ether-amide), anoligo(thioether-amide), an oligo(ester-amide), anoligo(thioester-amide), oligo(urea-amide), an oligo(ether-thioether), anoligo(ether-ester), an oligo(ether-thioester), an oligo ether-urea), anoligo(thioether-ester), an oligo(thioether-thioester), anoligo(thioether-urea), an oligo(ester-thioester), an oligo(ester-urea),and an oligo(thioester-urea), wherein L¹ is optionally substituted withone or more substituents independently selected from —OH, —OCH₃, —COOH,—COOCH₃, —NH₂, and —NHC(NH)NH₂; X³ is selected from an amide bond, anester bond, an ether, and an amine; R^(B) is a trivalent coupling group;X⁴ is selected from an amide bond, an ether bond, a thioether bond, anester bond, a thioester bond, a urea bridge, an amine bond, a linkinggroup of the formula:

wherein the amide bond marked by

is formed with the chelating group, and the other bond marked by

is bound to R^(B), and a linking group of the formula:

wherein the bond marked by

at the carbonyl end is formed with the chelating group, and the otherbond marked by

is bound to R^(B); R^(CH) is a chelating group containing a chelatednonradioactive cation.
 11. The conjugate in accordance with claim 10,wherein —X¹-L¹-X²— represents one of the following structures (L-1) and(L-2):—NH—C(O)—R⁶—C(O)—NH—R⁷—NH—C(O)—  (L-1)—C(O)—NH—R⁸—NH—C(O)—R⁹—C(O)—NH—R¹⁰—NH—C(O)—  (L-2) wherein R⁶ to R¹⁰ areindependently selected from C2 to C10 alkylene, which alkylene groupsmay each be substituted by one or more substituents independentlyselected from —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, and —NHC(NH)NH₂ orwherein —X¹-L¹-X²— represents one of the following structures (L-3) and(L-4):—NH—C(O)—R¹¹—C(O)—NH—R¹²—CH(COOH)—NH—C(O)—  (L-3)—C(O)—NH—CH(COOH)—R¹³—NH—C(O)—R¹⁴—C(O)—NH—R¹⁵—CH(COOH)—NH—C(O)—  (L-4)wherein R¹¹ to R¹⁵ are independently selected from C2 to C8 alkylene.12. The conjugate in accordance with claim 10 or claim 11, wherein R^(B)has the structure represented by formula (IV):

wherein: A is selected from N, CR¹⁶, wherein R¹⁶ is H or C1-C6 alkyl,and a 5 to 7 membered carbocyclic or heterocyclic group; the bond markedby

at (CH₂)_(a) is formed with X², and a is an integer of 0 to 4; the bondmarked by

at (CH₂)_(b) is formed with X³, and b is an integer of 0 to 4; and thebond marked by

at (CH₂)_(c) is formed with X⁴, and c is an integer of 0 to
 4. 13. Theconjugate in accordance with any one of claims 10 to 12, which is acompound of formula (IIIa):

or a pharmaceutically acceptable salt thereof, wherein: m is an integerof 2 to 6; n is an integer of 2 to 6; b is an integer of 0 to 4; c is aninteger of 0 to 4; R^(1L) is CH₂, NH or O; R^(3L) is CH₂, NH or O;R^(2L) is C or P(OH); X¹ is selected from an amide bond, an ether bond,a thioether bond, an ester bond, a thioester bond, an urea bridge, andan amine bond; L¹ is a divalent linking group with a structure selectedfrom an oligoamide, an oligoether, an oligothioether, an oligoester, anoligothioester, an oligourea, an oligo(ether-amide), anoligo(thioether-amide), an oligo(ester-amide), anoligo(thioester-amide), oligo(urea-amide), an oligo(ether-thioether), anoligo(ether-ester), an oligo(ether-thioester), an oligo ether-urea), anoligo(thioether-ester), an oligo(thioether-thioester), anoligo(thioether-urea), an oligo(ester-thioester), an oligo(ester-urea),and an oligo(thioester-urea), wherein L¹ is optionally substituted withone or more substitutents independently selected from —OH, —OCH₃, —COOH,—COOCH₃, —NH₂, and —NHC(NH)NH₂; X⁴ is selected from an amide bond, anether bond, a thioether bond, an ester bond, a thioester bond, a ureabridge, an amine bond, a linking group of the formula:

wherein the amide bond marked by

is formed with the chelating group, and a linking group of the formula:

wherein the bond marked by

at the carbonyl end is formed with the chelating group; and R^(CH) is achelating group containing a chelated nonradioactive cation.
 14. Theconjugate in accordance with any one of claims 1 to 13 wherein theconjugate is a compound selected from the group consisting of:

and pharmaceutically acceptable salts and individual isomers thereof,containing a chelated nonradioactive Gallium cation and wherein thefluorine atom is ¹⁸F.
 15. The compound according to claim 14, whereinthe compound is:

or a pharmaceutically acceptable salt thereof.
 16. A pharmaceutical ordiagnostic composition comprising or consisting of one or moreconjugates or compounds in accordance with any one of claims 1 to 15.17. A conjugate, compound or composition in accordance with any one ofclaims 1 to 14 for use as a cancer diagnostic or imaging agent.
 18. Amethod of imaging and/or diagnosing cancer comprising administering aconjugate, compound or composition according to any one of claims 1 to17 to a patient in need thereof.
 19. A conjugate, compound orcomposition in accordance with any one of claims 1 to 16 for use in thetreatment of cancer.
 20. A conjugate, compound or composition inaccordance with any one of claims 1 to 17 for the diagnosis, imaging orprevention of neoangiogenesis/angiogenesis.
 21. A conjugate, compound orcomposition in accordance with any one of claims 1 to 17 for use as acancer diagnostic or imaging agent or for use in the imaging of cancerwherein the cancer is prostate, breast, lung, colorectal or renal cellcarcinoma.